Use of oxygenated cholesterol sulfates for treating neurological conditions, neurodegenerative diseases, and addiction

ABSTRACT

Aspects of the present disclosure include methods for treating at least one of depression, neurodegenerative disease, multiple sclerosis, Parkinson&#39;s disease, spinocerebellar degeneration, Friedreich ataxia, ataxia-telangiectasia, progressive supranuclear palsy, Huntington&#39;s disease, striatonigral degeneration, olivopontocerebellar atrophy, Shy-Drager syndrome, schizophrenia, schizoaffective disorder, manic-depression (bipolar) disorder, disturbed or abnormal circadian entrainment, childhood Alice in Wonderland syndrome, childhood acute cerebellar ataxia, and Alzheimer&#39;s disease. In some instances, methods include treating an addiction to a drug, such as alcohol addiction, cocaine addiction or amphetamine addiction. In practicing the subject methods, an effective amount of at least one compound selected from 25-hydroxycholesterol-3-sulfate (25HC3S), 25-hydroxycholesterol-disulfate (25HCDS), 27-hydroxycholesterol-3-sulfate (27HC3S), 27-hydroxycholesterol-disulfate (27HCDS), 24-hydroxycholesterol-3-sulfate (24HC3S), 24-hydroxycholesterol-disulfate (24HCDS), and 24,25-epoxycholesterol-3-sulfate, or salt thereof is administered to the subject.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 63/044,631, filed Jun. 26, 2020, ProvisionalApplication No. 63/127,905, filed Dec. 18, 2020, Provisional ApplicationNo. 63/141,382, filed Jan. 25, 2021, Provisional Application No.63/146,559, filed Feb. 5, 2021, Provisional Application No. 63/146,563,filed Feb. 5, 2021, Provisional Application No. 63/146,565, filed Feb.5, 2021, Provisional Application No. 63/146,566, filed Feb. 5, 2021,Provisional Application No. 63/146,568, filed Feb. 5, 2021, ProvisionalApplication No. 63/149,977, filed Feb. 16, 2021, Provisional ApplicationNo. 63/149,993, filed Feb. 16, 2021, the disclosures of which areexpressly incorporated by reference herein in their entireties.

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

The invention was made, in part, with government support under VA MeritReview Grant, Grant No. 1I01BX003656 awarded by Veterans Affairs. Thegovernment has certain rights in the invention.

INTRODUCTION

Oxysterols have long been believed to be ligands of nuclear receptorssuch as liver×receptor (L×R), and they play an important role in lipidshomeostasis and immune system, where they are involved in bothtranscriptional and post-transcriptional mechanisms. Oxysterols are theoxidized form of cholesterol. In vivo, enzymatic transformation ofsterols to oxysterols is for biosynthesis of important biologicalproducts such as steroid hormones, bile acids, and vitamin D in cells,blood, and tissues. Oxysterols participate in many biological processesincluding cholesterol homeostasis, triglyceride metabolism, inflammatoryresponses, cell proliferation, platelet aggregation, and apoptosis. Theoxysterols have also been implicated in many diseases such as metabolicsyndrome and neurodegenerative diseases. Oxysterols can be sulfated bysulfotransferase 2B1b (SULT2B1b) at the 3-position of the ring A ofcholesterol to be oxysterol 3-sulfates including5-cholesten-3β-25-diol-3-sulphate (25HC3S),5-cholesten-3β-24-diol-3-sulphate (24HC3S),5-cholesten-3β-27-diol-3-sulphate (27HC3S) as well as Xol3S (cholesterol3-sulfate).

It has been shown previously that cholesterol metabolite,5-cholesten-3β-25-diol-3-sulphate, decreases lipid biosynthesis andincreases cholesterol secretion and degradation, and may be useful forthe treatment and prevention of hypercholesterolemia,hypertriglyceridemia, and conditions related to fat accumulation andinflammation (e.g., non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidneyinjury (AKI), psoriasis, and atherosclerosis). Oxysterols have also beenimplicated in several diseases such as metabolic syndrome. Oxysterolscan be sulfated, and the sulfated oxysterols act in different direction:they decrease lipid biosynthesis, suppress inflammatory responses, andpromote cell survival.

SUMMARY

The present disclosure provides methods for treating at least one ofdepression, neurodegenerative disease, multiple sclerosis, Parkinson'sdisease, spinocerebellar degeneration, Friedreich ataxia,ataxia-telangiectasia, progressive supranuclear palsy, Huntington'sdisease, striatonigral degeneration, olivopontocerebellar atrophy,Shy-Drager syndrome, schizophrenia, schizoaffective disorder,manic-depression (bipolar) disorder, disturbed or abnormal circadianentrainment, childhood Alice in Wonderland syndrome, childhood acutecerebellar ataxia, and Alzheimer's disease. In some instances, methodsinclude treating an addiction, such as alcohol addiction, cocaineaddiction, or amphetamine addiction. In practicing the subject methods,an effective amount of at least one oxysterol active agent compoundselected from 25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and 24,25-epoxycholesterolsulfate, or salt thereof is administered to the subject.

Aspects of the disclosure include:

1. A method of treating at least one of depression, neurodegenerativedisease, multiple sclerosis, Parkinson's disease, spinocerebellardegeneration, Friedreich ataxia, ataxia-telangiectasia, progressivesupranuclear palsy, Huntington's disease, striatonigral degeneration,olivopontocerebellar atrophy, Shy-Drager syndrome, schizophrenia,schizoaffective disorder, manic-depression (bipolar) disorder, disturbedor abnormal circadian entrainment, childhood Alice in Wonderlandsyndrome, childhood acute cerebellar ataxia, and Alzheimer's disease, ina subject in need thereof, comprising:

administering to the subject an effective amount of at least onecompound selected from 25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate, or salt thereof.

2. The method of aspect 1, wherein the method comprises administering tothe subject an effective amount of 25-hydroxycholesterol-3-sulfate(25HC3S) or salt thereof.3. The method of aspect 1, wherein the method comprises administering tothe subject an effective amount of 25-hydroxycholesterol-disulfate(25HCDS) or salt thereof.4. The method of aspect 1, wherein the method comprises administering tothe subject an effective amount of 27-hydroxycholesterol-3-sulfate(27HC3S) or salt thereof.5. The method of aspect 1, wherein the method comprises administering tothe subject an effective amount of 27-hydroxycholesterol-disulfate(27HCDS) or salt thereof.6. The method of aspect 1, wherein the method comprises administering tothe subject an effective amount of 24-hydroxycholesterol-3-sulfate(24HC3S) or salt thereof.7. The method of aspect 1, wherein the method comprises administering tothe subject an effective amount of 24-hydroxycholesterol-disulfate(24HCDS) or salt thereof.8. The method of aspect 1, wherein the method comprises administering tothe subject an effective amount of 24,25-epoxycholesterol-3-sulfate orsalt thereof.9. The method of any one of aspects 1 to 8, wherein the at least onecompound is administered in an amount ranging from 0.001 mg/kg/day to100 mg/kg/day.10. The method of any one of aspects 1 to 9, wherein the at least onecompound is administered in an amount ranging from 0.1 mg/kg to 100mg/kg, based on body mass of the subject.11. The method of any one of aspects 1 to 9, wherein the at least onecompound is administered in an amount ranging from 1 mg/kg to 10 mg/kg,based on body mass of the subject.12. The method of any one of aspects 1 to 11, wherein the administeringis performed from once to 3 times per day.13. The method of any one of aspects 1 to 12, wherein the administeringcomprises at least one of oral administration, enteric administration,sublingual administration, transdermal administration, intravenousadministration, peritoneal administration, parenteral administration,administration by injection, subcutaneous injection, and intramuscularinjection.14. The method of any one of aspects 1 to 13, wherein the administeringcomprises administering a pharmaceutical composition comprising the atleast one compound and a physiologically acceptable excipient, diluent,or carrier.15. The method of aspect 14, wherein the pharmaceutical composition isformulated in unit dosage form.16. The method of any one of aspects 14 or 15, wherein thepharmaceutical composition is in solid form.17. The method of any one of aspects 14 to 16, wherein thepharmaceutical composition is in the form of a powder, a tablet, acapsule, or a lozenge.18. The method of any one of aspects 14 to 17, wherein thepharmaceutical composition comprises the at least one compound infreeze-dried form together with a bulking agent.19. The method of any one of aspects 14 to 18, wherein thepharmaceutical composition is in a sealed vial, ampoule, syringe, orbag.20. The method of aspect 14 or 15, wherein the pharmaceuticalcomposition comprises a carrier that is a liquid.21. The method of aspect 20, wherein the at least one compound issolubilized in the liquid or dispersed in the liquid.22. The method of any one of aspects 20 and 21, wherein the liquid isaqueous.23. The method of any one of aspects 20 to 22, wherein the liquid issterile water for injections or phosphate-buffered saline.24. The method of any one of aspects 14 and 20 to 23, wherein thepharmaceutical composition is in a sealed vial, ampoule, syringe, orbag.25. Use of at least one compound selected from25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate, or salt thereof for the manufacture ofa medicament for treating at least one of depression, neurodegenerativedisease, multiple sclerosis, Parkinson's disease, spinocerebellardegeneration, Friedreich ataxia, ataxia-telangiectasia, progressivesupranuclear palsy, Huntington's disease, striatonigral degeneration,olivopontocerebellar atrophy, Shy-Drager syndrome, schizophrenia,schizoaffective disorder, manic-depression (bipolar) disorder, disturbedor abnormal circadian entrainment, childhood Alice in Wonderlandsyndrome, childhood acute cerebellar ataxia, and Alzheimer's disease, ina subject in need thereof.26. A method of treating an addiction in a subject in need thereof, themethod comprising administering to the subject at least one compoundselected from 25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate, or salt thereof in an amount effectiveto reduce the addiction.27. The method of aspect 26, wherein the method comprises administeringto the subject an effective amount of 25-hydroxycholesterol-3-sulfate(25HC3S) or salt thereof.28. The method of aspect 26, wherein the method comprises administeringto the subject an effective amount of 25-hydroxycholesterol-disulfate(25HCDS) or salt thereof.29. The method of aspect 26, wherein the method comprises administeringto the subject an effective amount of 27-hydroxycholesterol-3-sulfate(27HC3S) or salt thereof.30. The method of aspect 26, wherein the method comprises administeringto the subject an effective amount of 27-hydroxycholesterol-disulfate(27HCDS) or salt thereof.31. The method of aspect 26, wherein the method comprises administeringto the subject an effective amount of 24-hydroxycholesterol-3-sulfate(24HC3S) or salt thereof.32. The method of aspect 26, wherein the method comprises administeringto the subject an effective amount of 24-hydroxycholesterol-disulfate(24HCDS) or salt thereof.33. The method of aspect 26, wherein the method comprises administeringto the subject an effective amount of 24,25-epoxycholesterol-3-sulfateor salt thereof.34. The method of any one of aspects 26 to 33, wherein the addiction isat least one of drug addiction and behavioral addiction.35. The method of any one of aspects 26 to 33, wherein the addictioncomprises drug addiction.36. The method of any one of aspects 26 to 33, wherein the addictioncomprises addiction to a drug comprising at least one of alcohol,opioid, cocaine, and amphetamine.37. The method of any one of aspects 35 to 36, wherein administering theat least compound is effective to reduce a physiological dependence onthe drug by the subject.38. The method of any one of aspects 35 to 37, wherein administering theat least compound is effective to reduce a psychological dependence onthe drug by the subject.39. The method of aspect 35 to 38, wherein administering the at leastcompound is effective to reduce cravings for the drug by the subject.40. The method of any one of aspects 26 to 39, wherein the at least onecompound is administered in an amount ranging from 0.001 mg/kg/day to100 mg/kg/day.41. The method of any one of aspects 26 to 40, wherein the at least onecompound is administered in an amount ranging from 0.1 mg/kg to 100mg/kg, based on body mass of the subject.42. The method of any one of aspects 26 to 41, wherein the at least onecompound is administered in an amount ranging from 1 mg/kg to 10 mg/kg,based on body mass of the subject.43. The method of any one of aspects 26 to 42, wherein the administeringis performed from once to 3 times per day.44. The method of any one of aspects 26 to 43, wherein the administeringcomprises at least one of oral administration, enteric administration,sublingual administration, transdermal administration, intravenousadministration, peritoneal administration, parenteral administration,administration by injection, subcutaneous injection, and intramuscularinjection.45. The method of any one of aspects 26 to 44, wherein the administeringcomprises administering a pharmaceutical composition comprising the atleast one compound and a physiologically acceptable excipient, diluent,or carrier.46. The method of aspect 45, wherein the pharmaceutical composition isformulated in unit dosage form.47. The method of any one of aspects 45 or 46, wherein thepharmaceutical composition is in solid form.48. The method of aspect 45 to 47, wherein the pharmaceuticalcomposition is in the form of a powder, a tablet, a capsule, or alozenge.49. The method of aspect 45 to 48, wherein the pharmaceuticalcomposition comprises the at least one compound in freeze-dried formtogether with a bulking agent.50. The method of aspect 45 to 49, wherein the pharmaceuticalcomposition is in a sealed vial, ampoule, syringe, or bag.51. The method of aspect 45, wherein the pharmaceutical compositioncomprises a carrier that is a liquid.52. The method of aspect 51, wherein the at least one compound issolubilized in the liquid or dispersed in the liquid.53. The method of any one of aspects 51 and 52, wherein the liquid isaqueous.54. The method of any one of aspects 51 to 53, wherein the liquid issterile water for injections or phosphate-buffered saline.55. The method of any one of aspects 51 to 54, wherein thepharmaceutical composition is in a sealed vial, ampoule, syringe, orbag.56. Use of at least one compound selected from25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate, or salt thereof for the manufacture ofa medicament for treating an addiction in a subject in need thereof.57. At least one compound selected from 25-hydroxycholesterol-3-sulfate(25HC3S), 25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate, or salt thereof, for use in a methodfor treating at least one of depression, neurodegenerative disease,multiple sclerosis, Parkinson's disease, spinocerebellar degeneration,Friedreich ataxia, ataxia-telangiectasia, progressive supranuclearpalsy, Huntington's disease, striatonigral degeneration,olivopontocerebellar atrophy, Shy-Drager syndrome, schizophrenia,schizoaffective disorder, manic-depression (bipolar) disorder, disturbedor abnormal circadian entrainment, childhood Alice in Wonderlandsyndrome, childhood acute cerebellar ataxia, Alzheimer's disease, and anaddiction.58. At least one compound for use of aspect 57, wherein the method is amethod according to any one of aspects 1 to 24 and 26 to 55.59. Use according to aspect 25 or 56 wherein said treating is a methodof treating according to any one of aspects 1 to 24 and 26 to 55.60. The method of any one of aspects 1 to 24, the use of any one ofaspect 25 or 59, or the at least one compound for use of aspect 57 or58, wherein said at least one of depression, neurodegenerative disease,multiple sclerosis, Parkinson's disease, spinocerebellar degeneration,Friedreich ataxia, ataxia-telangiectasia, progressive supranuclearpalsy, Huntington's disease, striatonigral degeneration,olivopontocerebellar atrophy, Shy-Drager syndrome, schizophrenia,schizoaffective disorder, manic-depression (bipolar) disorder, disturbedor abnormal circadian entrainment, childhood Alice in Wonderlandsyndrome, childhood acute cerebellar ataxia, and Alzheimer's disease isat least one of at least one of depression, neurodegenerative disease,multiple sclerosis, Parkinson's disease, spinocerebellar degeneration,Friedreich ataxia, ataxia-telangiectasia, progressive supranuclearpalsy, Huntington's disease, striatonigral degeneration,olivopontocerebellar atrophy, Shy-Drager syndrome, schizophrenia,disturbed or abnormal circadian entrainment, childhood Alice inWonderland syndrome, childhood acute cerebellar ataxia, and Alzheimer'sdisease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D. Synthesis and enzyme kinetic studies of Xol3S, 25HC3S, and27HC3S. The biosynthesis of Xol3S, 25HC3S, and 27HC3S in the cells isshown in FIG. 1A. HPLC profiles of purified 25HC3S; Xol3S; and 27HC3Sare shown in FIG. 1B. Effects of 25HC3S, 27HC3S, Xol3S, and theirprecursors, 25HC, 27HC, and cholesterol, on DNMT1/3a/3b activities. Theconcentration dependent, 0-0.001 M (10 points), effects of 25HC3S,Xol3S, and 27HC3S on the enzyme activities is shown in FIG. 1C.Comparison of 25HC with 25HC3S, cholesterol with Xol3S, and 27HC with27HC3S is shown in FIG. 1D.

FIGS. 2A-2F. Effects of 25HC3S on DNA methylation in hepatocytes byglobal methylation sequencing analysis. Huh-7 cells were cultured in HGmedia for 72 hours and treated with ethanol (vehicle) and 25 mM 25HC3Sin ethanol for 4 hours. The levels of global methylation were estimatedby LINE-1 assay. Four CpG sites in promoter regions of LINE-1 elementwere chosen as the target positions as shown in FIG. 2A. Detailed globalmethylation was measured by WGBS. Circos maps of DMR distribution inchromosomes is shown in FIG. 2B: the first circle shows the distributionof hypermethylation DMRs; the second shows transposable element (TE)density; and the third shows the distribution of hypomethylation DMRs.Venn diagrams of hypomethylated DMR-associated genes (DMGs) in 25HC3Sand Vehicle libraries under CG, CHG, and CHH contexts of whole genome(Up) and promoter regions (Low) are shown in FIG. 2C. KOBAS software wasused to test the statistical enrichment of DMR related genes in theKyoto Encyclopedia of Genes and Genomes (KEGG) pathways. DNA methylationlevels in different genomic functional regions of the whole genome inFIG. 2D, where the x-axis represents the different genomic regions (CGI,CGI-shore, promoter, UTR 5, exon, intron, UTR 3, and repeat), and they-axis represents the methylation levels in 25HC3S and vehicle librariesunder CG, CHG, and CHH contexts. High enrichment of hypomethylated DMRsin whole genome in KEGG pathways is shown in FIG. 2E. High enrichment ofhypomethylated DMRs in promoter regions in KEGG pathways is shown inFIG. 2F. The detailed KEGG pathways are shown in Table 1.3.

FIGS. 3A-3D. Expression of key genes related to signaling pathways.Huh-7 cells were cultured in HG media for 72 hours and treated with25HC3S at 6.25 μM, 12.5 μM, 25 μM, and 50 μM for 1 hour, 2 hours, 4hours, 6 hours, and 8 hours. Key Genes and their targeting genesexpression were determined by RT-PCR analysis. The expressions of DUSP8(Dual Specificity Phosphatases 8), DUSP7 (Dual Specificity Phosphatases7), and MAPK1 (Mitogen-activated protein kinase 1) in MAPK signalingpathway are shown in FIG. 3A; their target genes, CREB (cAMP responsiveelement binding protein), PRDX6 (peroxiredoxin 6), and BAD (BCL2Associated Agonist Of Cell Death) are shown in FIG. 3B; Key genes, CACNAfamily (calcium voltage-gated channel subunits), in calcium-AMK pathwayare shown in FIG. 3C; their targeting genes PGC1A (PPARG co-activator 1alpha), HMGR (3-hydroxy-3-methylglutaryl-CoA reductase), and FAS (fattyacid synthase) are shown in FIG. 3D.

FIGS. 4A-4F. Effect of 25HC3S on transcription levels in hepatocytes.HepG-2 cells were cultured in HG media and treated with 25 μM of 25HC3Sfor 2 hours, 4 hours, and 8 hours. The up-regulated genes (>1.6 fold)are shown in FIG. 4A. Enrichment of up-regulated genes (8 hours) to Geneontological (GO) groups are shown in FIG. 4B (NRAP: negative regulationof apoptotic process; NRPCD: negative regulation of programmed celldeath; RS: regulation of signaling; SP: regulation of phosphorylation;NRCD negative regulation of cell death; RES: response to stress; NRPP:negative regulation of protein phosphorylation; CRCS: cellular responseto chemical stimulus; NRP: negative regulation of phosphorylation; ST:signal transduction). Down-regulated genes (reduction>40%) are shown inFIG. 4C. Enrichment of down-regulated genes (8 hours) to GO groups areshown in FIG. 4D (CLMP: cellular lipid metabolic process; SBP: steroidbiosynthetic process; AMP: alcohol metabolic process; CMP: cholesterolmetabolic process; FAMP: fatty acid metabolic process; TBP: triglyceridebiosynthetic process; NLBP: neutral lipid biosynthetic process; ACMP:acyl-CoA metabolic process; OABP: organic acid biosynthetic process;BAMP: bile acid metabolic process). Heatmap of up-regulated genesrelated to this study is shown in FIG. 4E; down-regulated genes areshown in FIG. 4F.

FIG. 5 . Sulfation of 25HC as an epigenetic regulatory pathway. 25HC isan endogenous agonist of DNMT-1 that methylates CpG in promoter regionsand subsequently silences gene expression, resulting in cell death andlipogenesis. 25HC can be sulfated to 25HC3S, which acts as an endogenousligand and inhibits activities of DNMTs. 25HC3S demethylates ^(5m)CpG inpromoter regions, and successively increases gene expression. Theeminent pathways regulated by the sulfation of oxysterol are involved inenergy and lipids metabolisms, MAPK-ERK, and calcium-AMPK. 25HC3Ssignificantly increases Dual-specificity phosphatases (DUSPs) and CREBexpression, which activate MAPK/ERK pathway, including CREB, BAD, andERK, and subsequently regulate cell survival and death. 25HC3S decreaseslipid biosynthesis and reduces lipid accumulation by demethylating^(5m)CpG in promoter regions, increasing expression of key genesinvolved in calcium channels and AMPK, and activating correspondingsignaling pathways, which result in increased oxidation of free fattyacids (FFA), and decreased biosynthesis of cholesterol and FFA. Theglobal regulation by sulfation of oxysterol suggests the physiologicaland pathophysiological significance of this regulatory mechanism.

FIG. 6 . Mechanisms of sulfation and metabolic pathways of oxysterolsulfates.

FIG. 7 . Regulatory pathway of oxysterol sulfation.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides methods for treating at least one ofdepression, neurodegenerative disease, multiple sclerosis, Parkinson'sdisease, spinocerebellar degeneration, Friedreich ataxia,ataxia-telangiectasia, progressive supranuclear palsy, Huntington'sdisease, striatonigral degeneration, olivopontocerebellar atrophy,Shy-Drager syndrome, schizophrenia, schizoaffective disorder,manic-depression (bipolar) disorder, disturbed or abnormal circadianentrainment, childhood Alice in Wonderland syndrome, childhood acutecerebellar ataxia, and Alzheimer's disease. For instance, the presentdisclosure provides methods for treating at least one of depression,neurodegenerative disease, multiple sclerosis, Parkinson's disease,spinocerebellar degeneration, Friedreich ataxia, ataxia-telangiectasia,progressive supranuclear palsy, Huntington's disease, striatonigraldegeneration, olivopontocerebellar atrophy, Shy-Drager syndrome,schizophrenia, disturbed or abnormal circadian entrainment, childhoodAlice in Wonderland syndrome, childhood acute cerebellar ataxia, andAlzheimer's disease.

In some instances, methods include treating an addiction, such asalcohol addiction, cocaine addiction, or amphetamine addiction.

In practicing the subject methods, one or more oxysterol active agentcompound selected from 25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate (24,25-EC3S), or salt thereof isadministered to a subject (e.g., human subject). As described herein,the compound 25-hydroxycholesterol-3-sulfate (25HC3S) refers to acompound having the chemical structure:

The compound 25-hydroxycholesterol-disulfate (25HCDS) refers to acompound having the chemical structure:

The compound 27-hydroxycholesterol-3-sulfate (27HC3S) refers to acompound having the chemical structure:

The compound 27-hydroxycholesterol-disulfate (27HCDS) refers to acompound having the chemical structure:

The compound 24-hydroxycholesterol-3-sulfate (24HC3S) refers to acompound having the chemical structure:

The compound 24-hydroxycholesterol-disulfate (24HCDS) refers to acompound having the chemical structure:

The compound 24,25-epoxycholesterol-3-sulfate (24, 25-EC3S) refers to acompound having the chemical structure:

Oxysterols according to certain instances, can be sulfated bysulfotransferase 2B1b (SULT2B1b) at the 3-position of the ring A ofcholesterol to be oxysterol 3-sulfates including 25HC3S, 24HC3S, 27HC3Sas well as Xol3S (cholesterol 3-sulfate) as summarized in FIG. 6 . Theoxysterol sulfate can be further sulfated by sulfotransferase 2A1(SULT2A1) to be oxysterol disulfates. For example, 25-hydroxycholesterol3-sulfate (25HC3S) can be further sulfated by SULT2A1 to 5-cholesten-3β,25-diol-disulfate (25HCDS). 25HC3S and 25HCDS are the only oxysterolsulfates that have been identified in vivo in hepatocyte nuclei while27HC3S in human sera and 24HC3S in urine. 25HC3S and 25HCDS are alsopotent regulators but function in a different direction from theirprecursor 25HC.

Cholesterol can be hydroxylated by CYP27A1 to 25HC or 27HC in themitochondria, and hydroxylated to 25HC by CYP3A4, or by cholesterol25-hydroxylase (CH25HL) in endoplasmic reticulum. Cholesterol can alsobe hydroxylated by cholesterol 24-hydroxylase to 24HC in brain tissue.This cholesterol precursor can also be used for synthesis of desmosterolvia a shunt of the mevalonate pathway. The desmosterol can be oxygenatedby CYP46A1 to form 24, 25-epoxycholesterol (24,25EC). 25HC, 27HC, 24HC,and cholesterol can be subsequently sulfated at the *position bySULT2B1b to form 25HC3S, 27HC3S, 24HC3S, and Xol3S, respectively. 24,25EC can be sulfated to be 24, 25EC3S.

While not being bound by theory, the function of 25HC and 25HC3S inglobal regulation indicates that they are epigenetic regulators.Methylation at position 5 of cytosine (5-methylcytosine, ^(5m)C) in DNApromoter regions is an important epigenetic modification that regulatesgene expression and other functions of the genome. Cytosine methylationof CpG in promoter regions is inversely correlated with transcriptionalactivity of associated genes as it causes chromatin condensation andgene silencing. Dysregulation of CpG methylation and gene expressionaffect metabolism, tissue function, and the metabolic state. Cytosinemethylation is catalyzed by DNA methyltransferases (DNMT-1, 3a/3b),which in some cases play a role in the regulation of DNAmethylation/demethylation. 25HC and 25HC3S are ligands of DNAmethyltranferase-1 (DNMT-1). In some cases, the oxysterol active agentcompounds described herein are cellular regulatory molecules thatepigenetically regulate lipid metabolism, cell survival/death, andinflammatory responses via DNA CpG methylation and ^(5m)CpGdemethylation. In some cases, high glucose incubation increases CpGmethylation in promoter regions via increasing nuclear 25HC levels,which silences key gene expressions involved in PI3K-Akt, cAMP, NAFLD,Type II Diabetes Mellitus, and Insulin Secretion signaling pathways. Incertain cases, oxysterol active agent compounds disclosed herein (e.g.,25HC3S) de-methylates ^(5m)CpG in these promoter regions, increases geneexpression, and up-regulates these signaling pathways. In some cases,the oxysterol active agent compound regulates the signaling pathways inan opposite direction from precursor 25HC. In some cases, the one ormore oxysterol active agent compounds regulate cell signaling pathwaysin response to stress responses. In certain cases, the one or moreoxysterol active agent compounds affect protein phosphorylation,inositol phosphorylation, and sphingosine phosphorylation in regulatingcellular functions. In certain cases, the one or more oxysterol activeagent compounds regulate gene expression at transcriptional levels. Anillustrative mechanism is depicted in FIG. 7 . The one or more oxysterolactive agent compounds (e.g., 25HC3S) in certain cases decrease lipidaccumulation, anti-inflammatory response, and anti-apoptosis byincreasing gene expression through demethylation of ^(5m)CpG in promoterregions of the key genes involved in MAPK-ERK and Calcium-AMPK signalingpathways, such as CREB5 (CAMP Responsive Element Binding Protein 5), BAD(BCL2 Associated Agonist of Cell Death), and ERK (Mitogen-activatedprotein kinase 1).

In some cases, the term “treat” is used herein to refer to administeringat least one oxysterol active agent compound selected from25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate, or salt thereof to a human subjectthat: (1) already exhibits at least one symptom of depression,neurodegenerative disease, multiple sclerosis, Parkinson's disease,spinocerebellar degeneration, Friedreich ataxia, ataxia-telangiectasia,progressive supranuclear palsy, Huntington's disease, striatonigraldegeneration, olivopontocerebellar atrophy, Shy-Drager syndrome,schizophrenia, schizoaffective disorder, manic-depression (bipolar)disorder, disturbed or abnormal circadian entrainment, childhood Alicein Wonderland syndrome, childhood acute cerebellar ataxia, orAlzheimer's disease; and/or (2) is diagnosed as having depression,neurodegenerative disease, multiple sclerosis, Parkinson's disease,spinocerebellar degeneration, Friedreich ataxia, ataxia-telangiectasia,progressive supranuclear palsy, Huntington's disease, striatonigraldegeneration, olivopontocerebellar atrophy, Shy-Drager syndrome,schizophrenia, schizoaffective disorder, manic-depression (bipolar)disorder, disturbed or abnormal circadian entrainment, childhood Alicein Wonderland syndrome, childhood acute cerebellar ataxia, orAlzheimer's disease, such as by a trained clinical professional. In somecases, “treatment” involves the lessening or attenuation, or in someinstances, the complete eradication, of at least one symptom ofdepression, neurodegenerative disease, multiple sclerosis, Parkinson'sdisease, spinocerebellar degeneration, Friedreich ataxia,ataxia-telangiectasia, progressive supranuclear palsy, Huntington'sdisease, striatonigral degeneration, olivopontocerebellar atrophy,Shy-Drager syndrome, schizophrenia, schizoaffective disorder,manic-depression (bipolar) disorder, disturbed or abnormal circadianentrainment, childhood Alice in Wonderland syndrome, childhood acutecerebellar ataxia, or Alzheimer's disease that was present prior to orat the time of administration of the at least one oxysterol active agentcompound selected from 25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate, or salt thereof. In some cases,treatment according to the present disclosure is sufficient to improveclinical indicators in the subject. In certain instances, theimprovement in the clinical indicators in the subject is such that thesubject is considered to no longer have depression, neurodegenerativedisease, multiple sclerosis, Parkinson's disease, spinocerebellardegeneration, Friedreich ataxia, ataxia-telangiectasia, progressivesupranuclear palsy, Huntington's disease, striatonigral degeneration,olivopontocerebellar atrophy, Shy-Drager syndrome, schizophrenia,schizoaffective disorder, manic-depression (bipolar) disorder, disturbedor abnormal circadian entrainment, childhood Alice in Wonderlandsyndrome, childhood acute cerebellar ataxia, or Alzheimer's disease.

In some cases, “treatment” involves the lessening or attenuation, or insome instances, the complete eradication, of at least one symptom of anaddiction to a drug. In some instances, treating a subject includesadministering at least one oxysterol active agent compound selected from25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate, or salt thereof to a human subjectthat: (1) already exhibits at least one symptom of an addiction; and/or(2) is diagnosed as having an addiction. For example, treating a subjectmay include administering at least one oxysterol active agent compoundselected from 25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate, or salt thereof to a human subjectthat: (1) already exhibits at least one symptom of alcohol addiction,cocaine addiction, or amphetamine addiction; and/or (2) is diagnosed ashaving alcohol addiction, cocaine addiction, or amphetamine addiction.In some cases, treating addiction to the drug includes reducing aphysiological dependence on the drug by the subject, such as wherephysiological dependence on the drug is reduced by 5% or more, such asby 10% or more, such as by 20% or more, such as by 30% or more, such asby 40% or more, such as by 50% or more, such as by 60% or more, such asby 70% or more, such as by 80% or more, such as by 90% or more, such asby 95% or more, such as by 97% or more, such as by 99% or more, andincluding where the physiological dependence on the drug is completelyeliminated (i.e., reduced by 100%). In some cases, treating addiction tothe drug includes reducing a psychological dependence on the drug by thesubject, such as where psychological dependence on the drug is reducedby 5% or more, such as by 10% or more, such as by 20% or more, such asby 30% or more, such as by 40% or more, such as by 50% or more, such asby 60% or more, such as by 70% or more, such as by 80% or more, such asby 90% or more, such as by 95% or more, such as by 97% or more, such asby 99% or more, and including where the psychological dependence on thedrug is completely eliminated (i.e., reduced by 100%). In certain cases,reducing psychological dependence on the drug is characterized byreduced cravings for the drug by the subject. For example, the least oneoxysterol active agent compound selected from25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate, or salt thereof may be administered tothe subject in an amount sufficient to reduce cravings for the drug bythe subject by 5% or more, such as by 10% or more, such as by 20% ormore, such as by 30% or more, such as by 40% or more, such as by 50% ormore, such as by 60% or more, such as by 70% or more, such as by 80% ormore, such as by 90% or more, such as by 95% or more, such as by 97% ormore, such as by 99% or more, and including where all cravings (i.e.,reduced by 100%) for the drug by the subject are completely eliminated.

In practicing the subject methods, an effective amount of at least oneoxysterol active agent compound selected from25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate, or salt thereof is administered to thesubject. In some cases, the oxysterol active agent compound isadministered to the subject at a dosage of from 0.00001 mg/kg/day to 500mg/kg/day, such as from 0.00005 mg/kg/day to 450 mg/kg/day, such as from0.0001 mg/kg/day to 400 mg/kg/day, such as from 0.0005 mg/kg/day to 350mg/kg/day, such as from 0.001 mg/kg/day to 300 mg/kg/day, such as from0.005 mg/kg/day to 250 mg/kg/day, such as from 0.01 mg/kg/day to 200mg/kg/day, such as from 0.05 mg/kg/day to 150 mg/kg/day, and includingfrom 0.001 mg/kg/day to 100 mg/kg/day. In certain cases, the oxysterolactive agent compound is administered to the subject at a dosage of from0.001 mg/kg/day to 100 mg/kg/day. In certain cases, the oxysterol activeagent compound is administered to the subject at a dosage of from 0.1mg/kg/day to 100 mg/kg/day. In certain cases, the oxysterol active agentcompound is administered to the subject at a dosage of from 1 mg/kg/dayto 100 mg/kg/day.

In some cases, the amount of each daily dose of the at least oneoxysterol active agent compound, such as 25-hydroxycholesterol-3-sulfateor 25-hydroxycholesterol-3-sulfate sodium, administered to theindividual is from 0.5 mg to 5 mg, 5 mg to 10 mg, 10 mg to 15 mg, 15 mgto 20 mg, 20 mg to 25 mg, 20 mg to 50 mg, 25 mg to 50 mg, 50 mg to 75mg, 50 mg to 100 mg, 75 mg to 100 mg, 100 mg to 125 mg, 125 mg to 150mg, 150 mg to 175 mg, 175 mg to 200 mg, 200 mg to 225 mg, 225 mg to 250mg, 250 mg to 300 mg, 300 mg to 350 mg, 350 mg to 400 mg, 400 mg to 450mg, or 450 mg to 500 mg. In some cases, the amount of oxysterol activeagent compound in the effective amount administered to the individual(e.g., a unit dosage form) is in the range of from 0.5 mg to 500 mg,such as from 1 mg to 450 mg, such as from 2 mg to 400 mg, such as from 5mg to 300 mg, such as from 10 mg to 200 mg, or such as from 20 mg to 100mg.

The oxysterol active agent compound may be administered to the subjectonce per day or more, such as twice per day or more, such as three timesper day or more, and including four times per day or more. For instance,the oxysterol active agent compound may be administered twice a day,once a day, once every other day, once every three days, once a week, oronce a month. In some cases, the oxysterol active agent compound isadministered to the subject once per day. In some cases, the oxysterolactive agent compound is administered to the subject twice per day. Insome instances, the oxysterol active agent compound is administered tothe subject once or twice per day in a cycle for a duration of rangingfrom 1 day to 10 days, 1 day to 30 days, 7 days to 30 days, 7 days to 90days, 10 days to 180 days, or 30 days to 1 year, 30 days to 5 years, 90days to 5 years, or 1 year to 10 years. In some cases, the oxysterolactive agent compound is administered to the subject once per day for aduration of from 1 day to 30 days, such as once per day for a durationof from 1 day to 28 days, from 1 day to 21 days, from 7 days to 14 days.In some cases, the oxysterol active agent compound is administered tothe subject twice per day for a duration of from 1 day to 30 days, suchas twice per day for a duration of from 1 day to 28 days, from 1 day to21 days, from 7 days to 14 days. In some cases, the oxysterol activeagent compound is administered to the subject three times per day for aduration of from 1 day to 30 days, such as three times per day for aduration of from 1 day to 28 days, from 1 day to 21 days, from 7 days to14 days.

In some cases, the dosing is administered in cycles of administration ofthe oxysterol active agent compound. In some cases, the cycle is 1 dayor more, such as 2 days or more, such as 3 days or more, such as 4, daysor more, such as 5 days or more, such as 6 days or more, such as 7 daysor more, such as 14 days or more, such as 21 days or more, such as 28days or more, and in some instances the cycle is 30 days or more. Thecycles of drug administration may be repeated for 1, 2, 3, 4, 5, 6, 7,8, or more than 8 dosage cycles, for a total period of 6 months, 1 year,2 years, 3 years, or 4 years or more. The administration of eachpharmaceutical composition can be extended over an extended period oftime (such as during maintenance therapy), such as from a month up toseven years. In some cases, the oxysterol active agent compound may beadministered over a period of about any of 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months. In other cases, theoxysterol active agent compound is administered for the rest of thesubject's lifetime.

Implementation of the methods generally involves identifying (e.g.,diagnosing) patients suffering from or at risk of depression,neurodegenerative disease, multiple sclerosis, Parkinson's disease,spinocerebellar degeneration, Friedreich ataxia, ataxia-telangiectasia,progressive supranuclear palsy, Huntington's disease, striatonigraldegeneration, olivopontocerebellar atrophy, Shy-Drager syndrome,schizophrenia, schizoaffective disorder, manic-depression (bipolar)disorder, disturbed or abnormal circadian entrainment, childhood Alicein Wonderland syndrome, childhood acute cerebellar ataxia, Alzheimer'sdisease, or addiction (e.g., alcohol, cocaine, or amphetamineaddiction). The exact dosage to be administered may vary depending onthe age, gender, weight, and overall health status of the individualpatient, as well as the precise etiology of the disease. The dose willvary with the route of administration, the bioavailability, and theparticular formulation that is administered, as well as according to thenature of the malady that is being prevented or treated. Further, theeffective dose can vary depending upon factors such as gender, age, andother conditions of the patient, as well as the extent or progression ofthe disease condition being treated. In some cases, each dosage of theoxysterol active agent compound is administered to the subject overduration of from 0.1 hours to 12 hours (e.g., by intravenousadministration) such as from 0.5 hours to 10 hours, such as from 1 hourto 8 hours, and including over a duration of from 2 hours to 6 hours.

Administration may be oral or parenteral, including intravenously,intramuscularly, subcutaneously, intradermal injection, intraperitonealinjection, etc., or by other routes (e.g., transdermal, sublingual,rectal and buccal delivery, inhalation of an aerosol, intravaginally,intranasally, topically, as eye drops, via sprays, etc.). In certaincases, the oxysterol active agent compound is administered to thesubject by one or more of oral administration, enteric administration,sublingual administration, transdermal administration, intravenousadministration, peritoneal administration, parenteral administration,administration by injection, subcutaneous injection, and intramuscularinjection. The route of administration will depend on the nature or thecondition that is treated, e.g., on the type or degree of the disease,and whether the treatment is prophylactic or intended to effect a cure.Further, administration of the compound by any means may be carried outas a single mode of therapy, or in conjunction with other therapies andtreatment modalities, e.g., diet regimens, etc.

In some cases, the compositions are administered in conjunction withother treatment modalities such as various pain relief medications,anti-arthritis agents, chemotherapeutic agents, antibiotic agents,anti-neurodegeneration agents, anti-addiction agents, anti-psychoticagents, anti-depressants (e.g., selective serotonin reuptake inhibitors(SSRI), selective serotonin & norepinephrine inhibitors (SNRI),monoamine oxidase inhibitors (MAOI), and N-methyl-D-aspartate (NMDA)receptor antagonists), and the like, depending on the malady that isafflicting the subject. “In conjunction with” refers to bothadministration of a separate preparation of the one or more additionalagents, and also to inclusion of the one or more additional agents in acomposition of the present disclosure.

For example, the oxysterol active agent may be administered inconjunction with at least one of chlorpromazine (e.g., Thorazine®),fluphenazine (e.g., Proxlixin®), haloperidol (e.g., Haldol®),perphenazine (e.g., Trilafon®), thioridazine (e.g., Mellaril®),thiothixene (e.g., Navane®), trifluoperazine (e.g., Stelazine®),aripiprazole (e.g., Abilify®), aripiprazole lauroxil (e.g., Aristada®),asenaphine (e.g., Saphris®), brexpiprazole (e.g., Rexulti®), cariprazine(e.g., Vraylar®), clozapine (e.g., Clozaril®), iloperidone (e.g.,Fanapt®), lumateperone tosylate (e.g., Caplyta®), lurasidone (e.g.,Latuda®), olanzapine (e.g., Zyprexa®), paliperidone palmitate (e.g.,Invega Trinza®), quetiapine (e.g., Seroquel®), risperidone (e.g.,Risperdal®), ziprasidone (e.g., Geodon®), citalopram (e.g., Celexa®),escitalopram oxalate (e.g., Lexapro®), fluoxetine (e.g., Prozac®),fluvoxamine (e.g., Luvox®), paroxetine (e.g., Paxil®), sertraline (e.g.,Zoloft®), desvenlafaxine (e.g., Khedezla®), desvenlafaxine succinate(e.g., Pristiq®), duloxetine (e.g., Cymbalta®), levomilnacipran (e.g.,Fetzima®), venlafaxine (e.g., Effexor®), vortioxetine (e.g.,Trintellix®), vilazodone (e.g., Viibryd®), amitriptyline (e.g.,Elavil®), imipramine (e.g., Tofranil®), nortriptyline (e.g., Pamelor®),doxepin (e.g., Sinequan®), bupropion (e.g., Wellbutrin®), isocarboxazid(e.g., Marplan), phenelzine (e.g., Nardil®), selegiline (e.g., Emsam®),tranylcypromine (e.g., Parnate®), esketamine (e.g., Spravato®),aducanumab, cholinesterase inhibitors, donepezil, rivastigmine,galantamine, glutamate regulators, memantine, orexin receptorantagonist, and suvorexant, and salts thereof.

The oxysterol active agent compounds may be administered in the pureform or in a pharmaceutically acceptable formulation including suitableelixirs, binders, and the like (generally referred to a “carriers”) oras pharmaceutically acceptable salts (e.g., alkali metal salts such assodium, potassium, calcium, or lithium salts, ammonium, etc.) or othercomplexes. It should be understood that the pharmaceutically acceptableformulations include liquid and solid materials conventionally utilizedto prepare both injectable dosage forms and solid dosage forms such astablets and capsules and aerosolized dosage forms. In addition, theoxysterol active agent compounds may be formulated with aqueous or oilbased vehicles. Water may be used as the carrier for the preparation ofcompositions (e.g., injectable compositions), which may also includeconventional buffers and agents to render the composition isotonic.Other potential additives and other materials (preferably those whichare generally regarded as safe [GRAS]) include: colorants; flavorings;surfactants (TWEEN®, oleic acid, etc.); solvents, stabilizers, elixirs,and binders or encapsulants (lactose, liposomes, etc). Solid diluentsand excipients include lactose, starch, conventional disintegratingagents, coatings, and the like. Preservatives such as methyl paraben orbenzalkium chloride may also be used. Depending on the formulation, itis expected that the active component (at least one oxysterol activeagent) will be present at 1% to 99% of the composition and the vehicular“carrier” will constitute 1% to 99% of the composition. Thepharmaceutical compositions of the present disclosure may include anysuitable pharmaceutically acceptable additives or adjuncts to the extentthat they do not hinder or interfere with the therapeutic effect of theat least one oxysterol active agent compound. Additional suitable agentsthat may be co-administered or co-formulated also include other agents,including but not limited to: metabolites of the methionine and/orglutathione biosynthetic pathways such as S-adenosylhomocysteine (SAH),S-methylmethionine (SMM), cystine, betaine, etc., or various formsand/or salts thereof, e.g., acetylcysteine (e.g., intravenousN-acetylcysteine), various neutraceuticals, etc.

Pharmaceutical compositions may include one or more pharmaceuticallyacceptable carriers. Pharmaceutically acceptable excipients have beenamply described in a variety of publications, including, for example, A.Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20thedition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Formsand Drug Delivery Systems (1999) H. C. Ansel et al., eds 7th ed.,Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. PharmaceuticalAssoc. For example, the one or more excipients may include sucrose,starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calciumphosphate, or calcium carbonate, a binder (e.g., cellulose,methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone,polyvinylpyrrolidone, gelatin, gum arabic, poly(ethylene glycol),sucrose or starch), a disintegrator (e.g., starch,carboxymethylcellulose, hydroxypropyl starch, low substitutedhydroxypropylcellulose, sodium bicarbonate, calcium phosphate, orcalcium citrate), a lubricant (e.g., magnesium stearate, light anhydroussilicic acid, talc, or sodium lauryl sulfate), a flavoring agent (e.g.,citric acid, menthol, glycine, or orange powder), a preservative (e.g.,sodium benzoate, sodium bisulfite, methylparaben, or propylparaben), astabilizer (e.g., citric acid, sodium citrate, or acetic acid), asuspending agent (e.g., methylcellulose, polyvinylpyrrolidone, oraluminum stearate), a dispersing agent (e.g.,hydroxypropylmethylcellulose), a diluent (e.g., water), and base wax(e.g., cocoa butter, white petrolatum, or polyethylene glycol).

In some cases, compositions of interest include an aqueous buffer.Suitable aqueous buffers include, but are not limited to, acetate,succinate, citrate, and phosphate buffers varying in strengths from 5 mMto 100 mM. In some cases, the aqueous buffer includes reagents thatprovide for an isotonic solution. Such reagents include, but are notlimited to, sodium chloride; and sugars, e.g., mannitol, dextrose,sucrose, and the like. In some cases, the aqueous buffer furtherincludes a non-ionic surfactant such as polysorbate 20 or 80. In someinstances, compositions of interest further include a preservative.Suitable preservatives include, but are not limited to, a benzylalcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. Inmany cases, the composition is stored at about 4° C. Formulations mayalso be lyophilized, in which case they generally includecryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol,and the like. Lyophilized formulations can be stored over extendedperiods of time, even at ambient temperatures.

In some cases, compositions include other additives, such as lactose,mannitol, corn starch, or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch, orgelatins; with disintegrators, such as corn starch, potato starch, orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives, and flavoring agents.

Where the composition is formulated for injection, the compositions maybe formulated by dissolving, suspending, or emulsifying the oxysterolactive agent compound in an aqueous or nonaqueous solvent, such asvegetable or other similar oils, synthetic aliphatic acid glycerides,esters of higher aliphatic acids, or propylene glycol; and if desired,with conventional additives such as solubilizers, isotonic agents,suspending agents, emulsifying agents, stabilizers, and preservatives.

In some cases, methods according to the present disclosure are directedto treatment of a subject based on a cellular response when the subjectis administered the oxysterol active agent compound. In some instancesof the present disclosure, epigenetic modification plays a role in theregulation and coordination of gene expression. Methylation at position5 of cytosine (5-methylcytosine, 5mC) in DNA is an important epigeneticmodification that regulates gene expression among other functions of thegenome. While not being bound by theory, cytosine methylation of CpG inthe promoter region is inversely correlated with transcriptionalactivity of associated genes as it causes chromatin condensation andthus gene silencing. Dysregulation of CpG methylation and geneexpression can affect tissue function and metabolic state. Cytosinemethylation is catalyzed by DNA methyltransferase (DNMT-1, 3a/3b), whichalso functions in the regulation of DNA methylation.

The major epigenetic regulation includes DNA and histone methylation,demethylation, acetylation, and deacetylation. The enzymes involved inthe process are DNA and histone methyltransferases/demethylases, andacetyltransferases/deacetylases. In some cases, administering one ormore of the oxysterol active agent compounds is sufficient to act as anepigenetic modulator of one or more of DNMT1, DNMT3a, DNMT3b, GCN3(Giant congenital nevi), p300 (histone acetyl transferase), Pcaf (KAT2Blysine acetyltransferase 2B), HDAC1 (histone deacetylase 1), HDAC2(histone deacetylase 2), HDAC3 (histone deacetylase 3), HDAC6 (histonedeacetylase 6), HDAC10 (histone deacetylase 10), and KDM6B-JMJD3 (lysinedemethylase 6B), such as where 25HC3S, 27HC, and 27HC3S, or cholesterol(Xol) and cholesterol-3-sulfate (Xol3S) are their endogenous ligand(s)to one or more of DNMT1, DNMT3a, DNMT3b, GCN3 (Giant congenital nevi),p300 (histone acetyl transferase), Pcaf (KAT2B lysine acetyltransferase2B), HDAC1 (histone deacetylase 1), HDAC2 (histone deacetylase 2), HDAC3(histone deacetylase 3), HDAC6 (histone deacetylase 6), HDAC10 (histonedeacetylase 10), and KDM6B-JMJD3 (lysine demethylase 6B).

While not being bound by theory, in some cases the one or moreadministered oxysterol active agent compounds inhibits DNMT-1, 3a, and3b, which demethylated ^(5m)CpG in promoter regions, increased geneexpression and up-regulated master signaling pathways such as MAPK,Calcium, AMPK, and CREB signaling pathways. In certain cases, the one ormore oxysterol active agent compounds regulate cell signaling pathwaysat transcriptional levels in nuclei. In some cases, the one or moreoxysterol active agent compounds are administered in an amountsufficient to affect protein phosphorylation, inositol phosphorylation,and/or sphingosine phosphorylation in regulating cellular functions.

In some cases, the addition of one or more of the subject oxysterolactive agent compound to human hepatocytes is sufficient to reversemethylation induced by HG, increase hypomethylated CpG in promoterregions of the key genes and increase targeting gene expression. In somecases, while not being bound by theory, CpG demethylation by theoxysterol active agent compound is the mechanism for its function ofglobal regulation: decreasing lipid accumulation, anti-inflammatoryresponses, anti-oxidants, and anti-cell death.

The DUSP family is a subset of protein tyrosine phosphatases, many ofwhich dephosphorylate mitogen-activated protein kinases (MAPKs) andhence are referred to as MAPK phosphatase. DUSP8, a unique member ofDUSP family, plays an important role in signal transduction of thephosphorylation-mediated MAPK pathway, which regulates responses tooxidative stress and cell death signals in various human diseases. Insome cases, administering the one or more oxysterol active agentcompounds is sufficient to demethylate ^(5m)CpG in promoter regions ofDUSP genes, including DUSP8, DUSP1, and DUSP7, and their downstreamgenes, CREB5, PRDX, BAD, and ERK, and increase their expression. Whilenot being bound by theory, the transcribed proteins from these genes areresponsible for cell survival and proliferation. In certain cases, theeffects of the at least one oxysterol active agent compound on promotingcell survival/proliferation and alleviating oxidative stress occurthrough inhibiting DNMTs and increasing expression of the DUSP family,especially DUSP8 and their downstream elements.

In some cases, a method of treatment involves modulating at least onegene selected from ABCC4, AC005264.2, ADCY1, ADCY4, ADCY5, ADH6, ADRB,ADRB1, AFDN, AGTR1, AKAP12, AL671762.1, ALAD, ANKRD1, ANKRD43, ATF3,ATP1A3, BAD, BIRC3, C11orf96, CACNA1A, CACNA1C-AS1, CACNA1D, CACNA1H,CACNB2, CACNG8, CELSR2, CREB5, CTB-186G2.1, CXCL2, CYB5B, CYP24A1,CYP51A1, CYR61, DDIT3, DRD5P2, DUSP genes, DUSP8, DUSP1, DUSP7, CREB5,EDNRB, EDN1, EHHADH, ELOVL6, ERK, FABP1, FDFT1, FRMD3, FMC1, FSTL3,GABBR1, GABBR2, GADD45B, GIPR, GLI3, GNA11, GNAQ, GNAS, GRIN2A, GRIN2C,GRIN3B, HBEGF, HMGCR, HMGCS1, HRAS, HRH1, HSPA6, ICAM1, ID3, ID4, IDI1,IL8, IL11, ITPKB, KANK4, KLB, KLFS, KLLN, KRTAP3-1, MAP2K6, MAP4K1,MAP4K4, MAPK1, MAPK8, MAT1A, MAX, METTL7A, MVK, NAP1L5, NCMAP, NTF3,P2RY8, PAQR8, PAQR9, PCSK9, PDE4D, PDGFB, PLA2G12B, PLCD1, PLPPR1,PMAIP1, PNPLA3, POU2AF1, PPP1CB, PRDX, PRLR, PTCH1, RAB11FIP4, RALGPS1,RAPGEF2, RELA, RHOBTB1, ROCK2, SC4MOL, SCN1A, SEC16B, SERPINE1, SKIL,SLC8A3, SLCO2B1, SLCO4C1, SLC2A14, SOCS2, SORBS2, SPHK1, SPTLC3, SQLE,TABS, TCIM, TGFB3, THBS1, TMEM170B, TNS1, TNFSF10, TUBB8, UBASH3B, VAV2,VAV3 and ZNF385B.

In some cases, a method of treatment involves modulating at least onepathway selected from cAMP signaling pathway, cGMP-PKG signalingpathway, circadian entrainment, glutamatergic synapse, adrenergicsignaling in cardiomyocytes, gap junction, Type II diabetes mellitus,endocytosis, calcium signaling pathway, dilated cardiomyopathy, vascularsmooth muscle contraction, MAPK signaling pathway, cholinergic synapse,Rapl signaling pathway, dopaminergic synapse, Adherens junction,arrhythmogenic right ventricular cardiomyopathy, pathways in cancer,GnRH signaling pathway, oxytocin signaling pathway, transcriptionalmisregulation in cancer, estrogen signaling pathway, insulin secretion,retrograde endocannabinoid signaling, long-term depression, colorectalcancer, insulin signaling pathway, axon guidance, alcoholism, plateletactivation, amphetamine addiction, herpes simplex infection, tightjunction, thyroid hormone signaling pathway, acute myeloid leukemia,chronic myeloid leukemia, notch signaling pathway, and dorso-ventralaxis formation.

EXPERIMENTAL

The present invention will be further illustrated by way of thefollowing Examples. These Examples are non-limiting and do not restrictthe scope of the invention. Unless stated otherwise, all percentages,parts, etc. presented in the Examples are by weight. The followingexamples are put forth so as to provide those of ordinary skill in theart with a complete disclosure and description of how to make and usethe present invention, and are not intended to limit the scope of whatthe inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Celsius, and pressure is ator near atmospheric. By “average” is meant the arithmetic mean. Standardabbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl,picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa,amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s);i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,subcutaneous(ly); and the like.

Example 1 Abbreviations

25HC 25-Hydroxycholesterol 25HC3S 25-Hydroxycholesterol 3-Sulfate 27HC27-Hydroxycholesterol 27HC3S 27-Hydroxycholesterol 3-Sulfate ^(5m)C5-Methylcytosine BAD BCL2 associated agonist of cell death CaV1 Calciumvoltage-gated channel subunit alpha1 D, CACNA1D CaV2 Calciumvoltage-gated channel subunit alpha1 A, CACNA1A CaV3 Calciumvoltage-gated channel subunit alpha1 H, CACNA1H CREB cAMP responseelement-binding protein DMEM Eagle's minimal essential medium DMGsDifferential methylated gene DMRs Differential methylated regions DNMTsDNA methyltransferases DUSP Dual-specificity phosphatase FAS Fatty acidsynthase GCN3 Giant congenital nevi HDAC1 Histone deacetylase 1 HDAC10Histone deacetylase 10 HDAC2 Histone deacetylase 2 HDAC3 Histonedeacetylase 3 HDAC6 Histone deacetylase 6 HG High glucose HMGR3-hydroxy-3-methylglutaryl-coenzyme A reductase KDM6B-JMJD3 Lysinedemethylase 6B KEGG Kyoto Encyclopedia of Genes and Genomes LINE Longinterspersed nuclear element LPS Lipopolysaccharides LXRs Nuclear liveroxysterol receptors MAPK A mitogen-activated protein kinase NAFLDNon-alcoholic fatty liver diseases NFκB Nuclear factorkappa-light-chain-enhancer of activated B cells p300 Histone acetyltransferase Pcaf KAT2B lysine acetyltransferase 2B PGC-1α Ppargcoactivator 1 alpha PPARγ Peroxisome proliferator-activated receptorPRDX6 Peroxiredoxin 6 RT-PCR Reverse transcription-polymerase chainreaction SREBP Sterol regulatory element-binding protein WGBS Wholegenome bisulfite sequencing Xol Cholesterol Xol3S Cholesterol 3-Sulfate

Materials and Methods

Materials

Cell culture reagents and supplies were purchased from GIBCO BRL (GrandIsland, N.Y.); Huh-7 cells were obtained from American Type CultureCollection (Rockville, Md.). The reagents for real time RT-PCR were fromAB Applied Biosystems (Warrington, UK). The chemicals used in thisresearch were obtained from Sigma Chemical Co. (St. Louis, Mo.) orBio-Rad Laboratories (Hercules, Calif.). All solvents were obtained fromFisher (Fair Lawn, N.J.) otherwise indicated.

Cell Culture

Huh-7 and HepG-2 cells were cultured in DMEM media supplemented with 10%heat-inactivated fetal bovine serum (FBS), high glucose (HG, 4.5 g/L) at37° C. in a humidified atmosphere of 5% CO₂.

Extraction and Determination of DNA and mRNA Levels

After culturing Huh-7 cells in DMEM medium with HG for 72 hours followedby treating with 25 μM 25HC3S for 4 hours, genomic DNA from 5,000 cellswere extracted using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). Eachsample, 2 μg, was sent to EpigenDx, Inc. (Hopkinton, Mass.) for analysisof global methylation bisulfite sequencing. The same samples, 6 μg, weresent to Novogene Co., Ltd (Tianjin, China) for analysis of whole genomebisulfite sequencing (WGBS). Total RNA was isolated using the Promega SVtotal RNA isolation system (Madison, Wis.) with DNase treatment. Eachsample, 2 μg, was used for the first-strand cDNA synthesis asrecommended by the manufacturer (Invitrogen, Carlsbad, Calif.).Real-time RT-PCR was performed using SYBR Green as the indicator on ABI7500 Fast Real-Time PCR System (Applied Biosystems, Foster City,Calif.). Amplifications of β-actin or GAPDH were used as internalcontrols. Relative messenger RNA (mRNA) expression was quantified withthe comparative cycle threshold (Ct) method using the primer set shownin Table 1.1 and was expressed as 2^(−ΔΔCt).

TABLE 1.1 Primer Sequence for Real-time Polymerase Chain Reaction Genename Forward Sequence Reverse Sequence DUSP8 TCAGCTCCGTCAACATCTGCCGCGTGCTCTGGTCATAGA (SEQ ID NO: 1) (SEQ ID NO: 15) DUSP7ATATCCTCAATGTCACACCC ATCTTCTGCATCAGATAGGCC AA (SEQ ID NO: 2)(SEQ ID NO: 16) MAPK1 ATGGTGTGCTCTGCTTATGA TCTTTCATTTGCTCGATGGTTGTA (SEQ ID NO: 3) (SEQ ID NO: 17) CREB5 GCAACAAGTCATCCCAGCATAAGAATCGGATTCAGGTCTGTT AAT (SEQ ID NO: 4) (SEQ ID NO: 18) PRDX6TCAATAGACAGTGTTGAGGA CCCGATTCCTATCATCGATGAT CC (SEQ ID NO: 5)(SEQ ID NO: 19) BAD ATGTTCCAGATCCCAGAGTT ATGATGGCTGCTGCTGGTTTG (SEQ ID NO: 6) (SEQ ID NO: 20) CaV1 AACAACAAACCAGAAGTCAACTAAGAATGAAGAAAGCGCTCC CC (SEQ ID NO: 7) (SEQ ID NO: 21) CaV2CGCTTCGGAGACGAGATGC TGCGCCATTGACTGCTTGT (SEQ ID NO: 8) (SEQ ID NO: 22)CaV3 CATGCTGGTAATCATGATCA CGAAAATGAAGGCGTCAAAGG AC (SEQ ID NO: 9)(SEQ ID NO: 23) PGC1A CACCAGCCAACACTCAGCTA ACGTCTTTGTGGCTTTTGCT(SEQ ID NO: 10) (SEQ ID NO: 24) HMGR GTCATTCCAGCCAAGGTTGTGGGACCACTTGCTTCCATTA (SEQ ID NO: 11) (SEQ ID NO: 25) FASTGTGGACATGGTCACGGAC GGCATCAAACCTAGACAGGTC (SEQ ID NO: 12)(SEQ ID NO: 26) β- CATGTACGTTGCTATCCAGG CTCCTTAATGTCACGCACGAT ACTINC (SEQ ID NO: 13) (SEQ ID NO: 27) GAPDH CAATGACCCCTTCATTGACCTTGATTTTGGAGGGATCTCG (SEQ ID NO: 14) (SEQ ID NO: 28)

Chemical Synthesis and Characterization of Sterol Sulfates, 25HC3S,Xol3S, 27HC3S

5-Cholesten-3β, 25-diol 3-sulfate (25-Hydroxycholesterol 3-Sulfate,25HC3S); 5-cholesten-3β-ol, 3-sulfate (Cholesterol 3-Sulfate, Xol3S);5-cholesten-3β, 27-diol 3-sulfate (27-Hydroxycholesterol 3-Sulfate,27HC3S) were synthesized as previously described with mild modification.Briefly, a mixture of 25-hydroxycholesterol, cholesterol, or27-hydroxycholesterol (6.5 mg, 0.016 mmol) and triethylamine-sulfurtrioxide (3.5 mg, 0.019 mmol) was dissolved in dry pyridine (300 μl) andwas stirred at room temperature for 2 hours. The solvents wereevaporated at 40° C. under nitrogen gas stream, and the syrup was addedinto 2 ml of 50% acetonitrile (loading buffer). The products wereapplied to a 6 cc Oasis cartridge (Waters), which had been primed bymethanol (15 ml) and water (15 ml). The cartridge was successivelywashed with the loading buffer (15 ml), water (15 ml), methanol (15 ml),50% methanol (15 ml), 5% ammonia hydroxide in 10% methanol (15 ml), and5% ammonia hydroxide in 50% methanol (15 ml). The retained sulphatedsterol was eluted with 5% ammonia hydroxide in 80% methanol (10 ml),respectively. After dilution with 10 times volume of acetonitrile, thesolvents were evaporated to dryness under nitrogen gas stream, and thesterol sulphates were obtained in white powder form.

Enzyme Kinetic Study of 5-Cholesten-3β, 25-Diol 3-Sulfate

For the DNMT1 activity assay, the substrate solution, 0.001 mg/mlPoly(dI-dC):Poly(dI-dC) in 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mMEDTA, 5 mM DTT, 1 mM PMSF, 5% glycerol, 0.01% Brij35, 1% DMSO was used.For the DNMT3a/3b activity assay, 0.0075 mg/ml Lambda DNA in 50 mMTris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 5 mM DTT, 1 mM PMSF, 5%glycerol, 1% DMSO, was used. The indicated DNMT1, DNMT3a, or DNMT3b wasadded to the appropriate substrate solution and gently mixed. Amounts ofcholesterol (Xol), 25HC, 27HC, Xol3S, 25HC3S, or 27HC3S ranging from5.08E-09 to 0.0001 M in DMSO were added to the reaction mixture by usingAcoustic Technology (Echo 550, LabCyte Inc. Sunnyvale, Calif.). Themixtures were first incubated for 15 min, then ³H-SAM was added to thereaction mixture to initiate the reaction, and the mixture was incubatedfor 60 min at 30° C. Following incubation, the reaction mixture wasfinally transferred to filter-paper for detection of radioactivitycounts.

Analysis of Global Methylation, Long Interspersed Nucleotide Element 1(LINE-1) Assay

For global DNA methylation analysis, 500 ng of extracted genomic DNA wasbisulfite-treated using the EZ DNA Methylation kit (Zymo Research, Inc.,CA). PCR reaction and product purification were performed as per themanufacturer's protocol (GE Healthcare Life Sciences). The PCR products,10 μl, were sequenced by Pyrosequencing on the PSQ96 HS System followingthe manufacturer's instructions (Pyrosequencing, Qiagen). Themethylation status of each CpG site was determined individually as anartificial C/T SNP using QCpG software (Pyrosequencing, Qiagen). Themethylation level at each CpG site was calculated as the percentage ofthe methylated alleles divided by the sum of all methylated andunmethylated alleles. The mean methylation level was calculated usingmethylation levels of all measured CpG sites within the targeted regionof each gene. Each experiment included non-CpG cytosines as internalcontrols to detect incomplete bisulfite conversion of the input DNA. Inaddition, a series of unmethylated and methylated DNA were included ascontrols in each PCR assay. Furthermore, PCR bias testing was performedby mixing unmethylated control DNA with in vitro methylated DNA atdifferent ratios (0%, 5%, 10%, 25%, 50%, 75%, and 100%), followed bybisulfite modification, PCR, and Pyro-sequencing analysis.

Analysis of Whole Human Genome Bisulfite Sequencing (WGBS)

Each sample, 5.2 μg of genomic DNA spiked with 26 ng lambda DNA, wasfragmented by sonication to 200-300 bp with Covaris S220, followed byend repair and adenylation. Cytosine-methylated barcodes were ligated tosonicated DNA per the manufacturer's instructions. These DNA fragmentswere treated twice with bisulfite using EZ DNA Methylation-Gold™ Kit(Zymo Research) before the resulting single-strand DNA fragments werePCR amplified using KAPA HiFi Hot Start Uracil and Ready Mix (2×).Library concentration was quantified by Qubit® 2.0 Flurometer (LifeTechnologies, CA, USA) and quantitative PCR, and the insert size wasassayed on an Agilent Bioanalyzer 2100 system.

The library preparations were sequenced on an Illumina Hiseq 2500/4000or Novaseq platform and 125 bp/150 bp paired-end reads were generated.Image analysis and base calling were performed with Illumina CASAVApipeline. Trimmomatic (Trimmomatic-0.36) software was used for qualitycontrol. Bismark software (version 0.16.3; Krueger F, 2011) was used toperform alignments of bisulfite-treated reads to a reference genome(−×700—dovetail). DSS software (23) was used to identify differentiallymethylated regions (DMRs). KOBAS software was used to test thestatistical enrichment of DMR related genes in the Kyoto Encyclopedia ofGenes and Genomes (KEGG) pathways.

Transcriptional Profiling and Data Analysis

Total RNA was extracted and purified from HepG-2 cells using SV totalRNA isolation system (Promega, Madison, Wis.). cDNAs were prepared andanalyzed using GeneChip® Human Genome U133 Plus 2.0 Array, Affymetrix(Santa Clara, USA) as previously described with technical support fromShanghai Biotechnology Corporation. Direct target genes in the presentstudy were selected based on more than 2-fold of reduction together witharray detect signal more than 5 in both samples. Genes showing foldchanges greater than 2 and array-detected signals greater than 7 in atleast one sample were selected as differently expressed genes. DAVIDsoftware (https://david.ncifcrf.gov/conversion.jsp) was used to analyzeGO enrichment of differently expressed genes.

Results 25-hydroxycholesterol-3-sulfate (25HC3S) Specifically InactivateDNMT Activities

In order to study the effects of sterol sulfates on the epigeneticregulating targets, 25HS3S, Xol3S, and 27HC3S (FIG. 1A) were synthesizedand purified to more than 95% purity using triethylamine sulphatecomplex methods as shown in FIG. 1B. Results show that 25HC3Ssignificantly inhibits only DNMT-1, 3a, and 3b activities withIC₅₀=4.04, 3.03, and 9.05×10⁻⁶M, respectively (FIG. 1C, Left), while itsprecursor 25HC activates DNMT-1 activity by 8-fold with EC50=3.5×10⁻⁶ M(FIG. 1D, Left). As controls, Xol as well as Xol3S did not significantlyaffect enzymatic activities although Xol3S slightly inhibits DNMT3a withIC₅₀=8.2×10⁻⁵ M, which is most likely not physiologically significant(FIG. 1C, Middle). Compared with 25HC3S, 27HC3S did inhibit DNMTs withsimilar IC₅₀=3.58×10⁻⁶ M for DNMT1, 8.88×10⁻⁶ M for DNMT3a, and2.68×10⁻⁶ M for DNMT3b as shown in FIG. 1C. Right. In contrast, itsprecursor 27HC, was much less potent in activation of DNMT-1 withEC50=3.3×10⁻⁵ M and had no effect on other enzymes (FIG. 1D, Right). Incontrast to the 3 DNMTs, the 9 other epigenetic enzymes are not affectedby these oxysterols or sterol sulfates (data not shown). As a positivecontrol, S-adenosyl homocysteine (SAH) inhibited DNMT1 activity by 95%at 1 μM (data not shown), as previously reported. The resultsdemonstrated that both 25HC3S and 27HC3S are potent inhibitors of DNMTs.However, only 25HC3S has been discovered in vivo in human hepatocytenuclei: first found in concentration of 20 ug/g (˜40 uM) followingoverexpression of mitochondrial cholesterol delivery protein, StarD1.The kinetic study shows that the IC50s are between 1-10 uM.

25-hydroxycholesterol-3-sulfate (25HC3S) Decreases ^(5m)CpG Levels inGlobal Promoter Regions

Previous studies have shown that global DNA methylation and themethylation of specific genes are involved in adipogenesis, lipidmetabolism, and inflammation in visceral adipose tissues, which, inturn, are related to the specific etiology of metabolic syndrome. Tostudy the effects of 25HC3S on methylation status of ^(5m)CpG in globalpromoter regions, LINE-1 analysis was first performed to estimate globaldemethylation. Methylation usually occurs in repetitive elements, suchas LINE elements. There are ˜500,000 LINE elements and 750 millioncopies in total human genome. Human LINE-1 is a retro-transposableregion (promoter region) and has only 700,000 copies, which correlatesto ˜17% of the human genome. The specific sequence includes four CpGdinucleotides (Pos 1, 2, 3, and 4), which serve asmethylation/demethylation targets in LINE-1. As shown in FIG. 2A,culturing Huh-7 cells in high glucose media (HG), Pos 3 and Pos 4 hadhigher methylation, while all 4 Pos increased methylation afterculturing cells in ethanol control. Reduction of methylation(demethylation) at Pos1 (−5%), Pos3 (−10%), and Pos 4 (−5.6%) occurredafter incubating cells with 25HC3S for 4 hours. The results indicatethat 25HC3S significantly reduce ^(5m)CpG methylation in promoterregions induced by HG or ethanol.

Profiles of Whole Genome-Wide DNA Methylation in 25HC3S-Treated Humanhepatocytes

To understand the possible cellular functions of ^(5m)CpG demethylationin 25HC3S-treated Huh-7 cells, the cells were harvested for theconstruction of bisulfite-treated genomic DNA libraries. In 5 totalWGBS, there were 366 million (Vehicle) and 370 million (25HC3S-treated)raw reads generated from the two libraries by paired-end sequencing,respectively. Among clean reads, 360 million, from vehicle library, 77%(277 million) were uniquely mapped to the reference genome of “humanreference genome (hg38)”, while among 365 million clean reads from the25HC3S-treated library, 78% (286 million) were uniquely mapped to thereference genome, exhibiting an 10 average read depth of 22 and 20,respectively. In these two libraries, more than 80% of cytosine residueswere covered by at least ten reads in “human reference genome (hg38)”.The depth and density of the sequencing were enough for a high-qualitygenome-wide methylation analysis. Meanwhile, the efficiencies ofbisulfite conversion, represented by the lambda DNA to the libraries,were over 99%, providing reliable and accurate results for the WGBS(Table 1.2).

TABLE 1.2 Summary of The Whole Genome Bisulfite Sequencing Data CleanUniquely Uniquely BS Sample Raw Clean ratio mapped mapped Sites_numconversion name reads reads (%) reads rate (%) Sites_covgMean Covg10rate(%) Vehicle 366,129,468 360,798,889 90.30 276,588,428 76.66 19.6880.78 99.727 25HC3S 370,635,495 365,154,331 90.15 285,623,717 78.2221.09 83.17 99.795

CpG methylation and demethylation are well documented to related withgene expression. A total of about 7,136 differential methylated regions(DMRs) under CG context were identified as hypomethylated regionslocated in 1,106 genes (differential methylated genes, DMGs). In 97%(1,074) of the DMGs, the hypomethylated regions were identified in theirpromoters (FIG. 2B). The hypomethylated genes were highly enriched in 75KEGG pathways (p<0.05) (Table 1.3). The top 20 pathways (from the mostsignificance, p<10⁻⁹) were shown in FIGS. 2E and 2F. Among thesepathways, MAPK-ERK and calcium-cAMP signaling are believed as the masterpathways regulating cell survival, anti-oxidants, anti-apoptosis, energymetabolism, and lipid homeostasis. The pathways identified from wholegenome are shown in FIG. 2E, and those identified from promoter regionsare shown in FIG. 2F. Both sets of pathways, from whole genome or frompromoter regions, are very similar. All pathways identified frompromoter regions were hypomethylated without any hypermethylated CpGs intheir promoter regions, indicating up-regulated gene expressions.

DNA methylation levels in whole genome and differential methylatedregions (DMRs) are shown in FIG. 2B. To present the global DNAmethylation profiles of the two libraries, the uneven methylation levelsthroughout the chromosomes under CG, CHG (H represents adenosine orthymidine residues), and CHH contexts are shown in FIG. 2B. A total of6,923 differentially methylated genes (DMGs) were screened out among thetwo libraries. Moreover, 1,510 were 20 identified under CG context, 420under CHG context, and 3,359 under CHH context. 83 were identified underCG and CHG contexts, 481 under CG and CHH contexts, 793 under CHG andCHH contexts, while only 277 were identified under CG, CHG, and CHHcontexts. Furthermore 2,853 were identified as promoter regions, and1,413 were identified under CG context, 186 under CHG context, and 787under CHH context. For these DMGs, 59 were identified under CG and CHGcontexts, 46 under CG and CHH, 260 under CHG and CHH contexts, and only103 were identified under CG, CHG, and CHH contexts. While in theseDMGs, 80.93% (5,603) were identified as hypomethylated, and 37.55%(2,104) were identified as hypomethylated in promoter 5 regions (FIG.2C).

TABLE 1.3 Significant Enrichment KEGG Pathways of Hypomethylated DMGs inPromoter Region under CG Context Pathway name P-value cAMP signalingpathway 3.69E−07 cGMP-PKG signaling pathway 2.65E−05 Circadianentrainment 5.68E−05 Glutamatergic synapse 8.40E−05 Adrenergic signaling0.000108 in cardiomyocytes Gap junction 0.000227 Type II diabetesmellitus 0.000505 Endocytosis 0.000656 Calcium signaling pathway0.000656 Dilated cardiomyopathy 0.000656 Vascular smooth 0.000752 musclecontraction MAPK signaling pathway 0.000877 Cholinergic synapse 0.001123Rap1 signaling pathway 0.001123 Dopaminergic synapse 0.001194 Adherensjunction 0.001194 Arrhythmogenic right 0.001203 ventricularcardiomyopathy Pathways in cancer 0.001203 GnRH signaling pathway0.001679 Oxytocin signaling pathway 0.002196 Transcriptional 0.002515misregulation in cancer Estrogen signaling pathway 0.002932 Insulinsecretion 0.003045 Retrograde 0.003437 endocannabinoid signalingLong-term depression 0.003463 Colorectal cancer 0.004132 Insulinsignaling pathway 0.004549 Axon guidance 0.005282 Alcoholism 0.005377Platelet activation 0.006325 Amphetamine addiction 0.006325 Herpessimplex infection 0.006736 Tight junction 0.007081 Thyroid hormone0.007681 signaling pathway Acute myeloid leukemia 0.007681 Chronicmyeloid leukemia 0.008581 Notch signaling pathway 0.012054 Dorso-ventralaxis formation 0.013526 Cocaine addiction 0.014096 Viral carcinogenesis0.01429 Pancreatic cancer 0.015201 Inflammatory mediator 0.015802regulation of TRP channels Hippo signaling pathway 0.017014Melanogenesis 0.017221 Neurotrophin 0.018561 signaling pathwaySalmonella infection 0.018615 Chagas disease 0.01954 (Americantrypanosomiasis) Thyroid hormone synthesis 0.021071 HTLV-I infection0.021071 Prostate cancer 0.021071 GABAergic synapse 0.021665 Salivarysecretion 0.021665 Cell adhesion 0.022648 molecules (CAMs) Amoebiasis0.023054 Viral myocarditis 0.024448 Type I diabetes mellitus 0.024448Focal adhesion 0.024448 Ras signaling pathway 0.024601 Fructose andmannose 0.025877 metabolism One carbon pool by folate 0.027147Serotonergic synapse 0.027147 Endocrine and other factor- 0.029495regulated calcium reabsorption Hypertrophic 0.034694 cardiomyopathy(HCM) Long-term potentiation 0.036088 Ovarian steroidogenesis 0.036088Wnt signaling pathway 0.036088 Endometrial cancer 0.038405 AMPKsignaling pathway 0.040972 Fc epsilon RI 0.041787 signaling pathwayAllograft rejection 0.045211 Bile secretion 0.045703 Prolactin signalingpathway 0.045703 Chemokine signaling pathway 0.046416 Neuroactiveligand- 0.046416 receptor interaction Fc gamma R- 0.047019 mediatedphagocytosis

Previous report has shown that high glucose incubation (HG), an in vitromodel for study of NAFLD, induces lipid accumulation via increasing DNApromoter methylation signaling. It was noted that the hypermethylated^(5m)CpG in the promoter regions induced by HG were demethylated by25HC3S. 25HC3S demethylated ^(5m)CpG in promoter regions of 23 genes inMAPK signaling pathway (Table 1.4), 19 genes in Calcium pathway (Table1.5), and 28 genes in cAMP pathway (Table 1.6). No hypermethylated DMRwas found in the genes involved in the signaling pathways. Thechromosome and sequence location of the hypermethylated ^(5m)CpG by HGand the hypomethylated CpG by 25HC3S in promoter regions are compared inthe tables. It is observed that these genes are also involved in manyother KEGG pathways including insulin, Type II Diabetes Mellitus, andcGMP-PKG signaling pathways. The results indicate that the globalregulatory mechanisms of 25HC3S are through demethylation of ^(5m)CpG inpromoter regions of the key genes, such as the DUSP and Calcium channelfamilies, involved in MAPK-ERK and calcium-cAMP master signalingpathways.

DNA methylation levels generally show a varied distribution acrossdifferent functional regions of the genome. The methylation levels inthe CGI (CG island), CGI-shore (up to 2 k bp away from the CGI),promoter (upstream 2 k bp sequence from transcription starting site),5′untranslated region (UTR5), exon, intron, 3′untranslated region(UTR3), and repeat were 10 significant different between vehicle and25HC3S treated groups. It is interesting that 25HC3S treatment resultedin significantly higher hypomethylation levels than vehicle (FIG. 2D).In a total of 34,508 DMRs identified, 3,676 (1,549 hypermethylated and2,127 hypomethylated) were distributed in CGI, 2206 (627 hypermethylatedand 1,579 hypomethylated) in CGI-shore, 3,263 (1,213 hypermethylated and2,050 hypomethylated) in exon, 9,850 (2,340 hypermethylated and 15 7,510hypomethylated) in intron, 3,696 (1,187 hypermethylated and 2,509hypomethylated) in promoter, 8,956 (1,882 hypermethylated and 7,774hypomethylated) in repeat region, 61 (16 hypermethylated and 45hypomethylated) in TES elements, 452 (179 hypermethylated and 273hypomethylated) in TSS elements, 403 (123 hypermethylated and 280hypomethylated) in UTR3 regions, and 1245 (432 hypermethylated and 813hypomethylated) in UTR5 regions. In almost all 20 DMRs, CpGs aresignificantly more hypomethylated than hypermethylated. It has beenreported that CG methylation in promoter regions plays a key role insilencing gene expression.

In a total of 6,923 DMGs, the genes under CG context were highlyenriched in 120 KEGG pathways (69 hypomethylated and 51hypermethylated). The genes under CHG context were enriched in 48pathways (33 hypomethylated and 15 hypermethylated), while those underCHH context, enriched in 136 pathways (101 hypomethylated and 35hypermethylated). DMGs in promoter regions were highly enriched in 114(31 hypermethylated and 83 hypomethylated) pathways, of which 75 (0hypermethylated and 75 hypomethylated) under CG context, 13 (13hypermethylated and 0 hypomethylated) under CHG context, and 26 (18hypermethylated and 8 hypomethylated) under CHH context (Table 1.3).

TABLE 1.4 Demethylation of ^(5m)CpG in Promoter Regions of MAPKSignaling Genes (P = 0.00087) DMR location DMR in promoter region(Methylation %) Chro- 25HC3S- Gene name mosome Start End HGLG VehicleMAPK8 Chr10 48306510 48306561 −29.24 DUSP8 Chr11 1572973 1573032 −30.511573583 1573855 +41.72 MAX Chr14 65102273 65102358 −31.75 DUSP7 Chr3 52056416 52056485 −40.47 52056538 52056778 +31.75 NTF3 Chr12 54327965432882 −33.48 CACNA1D Chr3  53494583 53494705 −20.46 53493469 53494019+49.03 CACNA1H Chr16 1194928 1195015 −26.48 1194670 1195215 +41.26CACNA1A Chr19 13226635 13226711 −39.91 13238959 13239237  +9.51 MAPK1Chr22 21867450 21867512 −7.66 21867333 21867621 +20.15 HRAS Chr11 535071535127 −18.85 536242 537214 +42.8  PDGFB Chr22 39243967 39244084 −30.5139242292 39242477 +29.29 CACNA1C-AS1 Chr12 2691335 2691438 −20.97 CACNB2Chr10 18140401 18140547 −21.91 18340356 18341053 +47.13 MAP4K1 Chr1938596260 38596495 −34.3 RAPGEF2 Chr4  1.59E+08 1.59E+08 −36.05CTB-186G2.1 Chr19 38596260 38596495 −34.3 RELA Chr11 65661914 65662035−35.1 GADD45B Chr19 2474806 2474873 −28.6 AL671762.1 Chr6  3182801931828149 −27.17 CACNG8 Chr19 53961377 53961479 −31.18 MAP4K4 Chr2 1.02E+08 1.02E+08 −13.63 1.02E+08 1.02E+08 +17.93 TGFB3 Chr14 7598239575982557 −23.74Table 1.4—After culturing Huh-7 cells in DMEM medium with HG for 72hours followed by treating with ethanol (vehicle) and 25 μM 25HC3S for 4hours, genomic DNA from 5,000 cells were extracted using QIAamp DNA MiniKit (QIAGEN, Hilden, Germany). Each sample (6 μg) was used for analysisof the whole genome bisulfite sequencing (WGBS). The KEGG analysis showsthat the demethylated genes are involved in MAPK signaling pathway(p=0.00087). Of the 257 total genes in the MAPK signaling pathway, 23were demethylated by the 25HC3S treatment. Of these 23 genes, 10 werefound to be methylated by a HG environment (shown in bold). The firstcolumn represents the gene name, the second column (DMR location inpromoter region) shows the location of differential methylation regionin the chromosome, the third column (DMR (Methylation %)) shows themethylation rates by high glucose (HG) and demethylation rates inducedby 25HC3S.

TABLE 1.5 Demethylation of ^(5m)CpG in Promoter Regions of CalciumSignaling Genes (P = 0.00066) DMR location DMR in promoter region(Methylation %) Chro- 25HC3S- Gene name mosome Start End HGLG VehicleADCY4 Chr14 24334570 24334719 −18.63 24334404 24334719 +32.6  DRD5P2Chr1  1.43E+08 1.43E+08 −34.73 EDNRB Chr13 77919496 77919743 −34.09ADRB1 Chr10 1.14E+08 1.14E+08 −33.1 GRIN2A Chr16 10183302 10183414−19.63 10084383 10084724  +8.62 GNA11 Chr19 3094383 3094442 −32.663092574 3092876  +6.13 SPHK1 Chr17 76383180 76383291 −24.74 CACNA1C-AS1Chr12 2691335 2691438 −20.97 HRH1 Chr3  11154292 11154354 −25.48 ITPKBChr1  2.27E+08 2.27E+08 −25.12 2.27E+08 2.27E+08 +23.84 PLCD1 Chr3 38029863 38030016 −24.31 38029901 38030042 +26.02 GNAS Chr20 5888859858888893 −25.68 58888560 58888756 +23.2  AC005264.2 Chr19 31563923156457 −15.76 CACNA1D Chr3  53494583 53494705 −20.46 53493469 53494019+49.03 GNAQ Chr9  78032198 78032440 −11.92 GRIN2C Chr17 7486173974861853 −37.6 74854901 74855100 +36.66 SLC8A3 Chr14 70188747 70189089−28.51 70046033 70046320 +65.66 CACNA1H Chr16 1194928 1195015 −26.481194670 1195215 +41.26Table 1.5—Cells preparation and DNA methylation as described in Table1.1. The KEGG analysis shows that the demethylated genes are involved incalcium signaling pathway (P=0.00066). Of the 180 total genes in theCalcium signaling pathway, 19 were demethylated by the 25HC3S treatment.Of these 19 genes, 10 were found to be methylated by a HG environment(shown in bold). The first column represents the gene name, the secondcolumn (DMR location in promoter region) shows the location ofdifferential methylation region in the chromosome, the third column (DMR(Methylation %)) shows the methylation rates by high glucose (HG) anddemethylation rates induced by 25HC3S.

TABLE 1.6 Demethylation of ^(5m)CpG in Promoter Regions of cAMPSignaling Genes (P = 3.69E−07) DMR location DMR in promoter region(Methylation %) Chro- 25HC3S- Gene name mosome Start End HGLG VehicleMAPK8 Chr10 48306510 48306561 −29.24 PTCH1 Chr9  95508446 95508561−12.09 ADCY5 Chr3  1.23E+08 1.23E+08 −30.44 1.23E+08 1.23E+08 +15.8 GLI3 Chr7  42228171 42228223 −19.62 42184839 42185064  +8.27 PPP1CBChr2  28751716 28751798 −10.93 28793684 28794075  +7.5  GNAS Chr2058888598 58888893 −25.68 58888560 58888756 +23.2  CACNA1D Chr3  5349458353494705 −20.46 53493469 53494019 +49.03 GRIN2A Chr16 10183302 10183414−19.63 MAPK1 Chr22 21867450 21867512 −7.66 21867333 21867621 +20.15GABBR2 Chr9  98708549 98708610 −21.84 CREB5 Chr7  28489448 28489601−29.31 28776194 28776325 +27.34 GRIN3B Chr19 1000331 1000405 −34.67CACNA1C-AS1 Chr12 2691335 2691438 −20.97 VAV2 Chr9  1.34E+08 1.34E+08−33.11 1.34E+08 1.34E+08 +44.31 ABCC4 Chr13 95301634 95301736 −33.06PDE4D Chr5  59893498 59893718 −27.84 59215742 59215941 +39.66 ROCK2Chr2  11344628 11344733 −23.14 ADCY1 Chr7  45574500 45574591 −14.7645574683 45574877 +25.79 ADCY4 Chr14 24334570 24334719 −18.63 2433440424334719 +32.6  DRD5P2 Chr1  1.43E+08 1.43E+08 −34.73 ADRB1 Chr101.14E+08 1.14E+08 −33.1 GIPR Chr19 45668710 45668767 −10.04 4566907545669744 +43.62 RELA Chr11 65661914 65662035 −35.1 AFDN Chr6  1.68E+081.68E+08 −30.68 BAD Chr11 64286088 64286179 −42.33 ATP1A3 Chr19 4199913441999316 −15.75 GRIN2C Chr17 74861739 74861853 −37.64 74854901 74855100+36.66Table 1.6—Cells preparation and DNA methylation as described in Table1.1. The KEGG analysis shows that the demethylated genes aresignificantly involved in cAMP signaling pathway (P=3.69E-07). Of the200 total genes in the cAMP signaling pathway, 28 were demethylated bythe 25HC3S treatment. Of these 28 genes, 13 were found to be methylatedby a HG environment (shown in bold). The first column represents thegene name, the second column (DMR location in promoter region) shows thelocation of differential methylation region in the chromosome, the thirdcolumn (DMR (Methylation %)) shows the methylation rates by high glucose(HG) and demethylation rates induced by 25HC3S.

Relationship Between ^(5m)CpG Demethylation in Promoter Regions and GeneExpression: 25HC3S Decreases HG-Induced ^(5m)CpG Levels in PromoterRegions

To explore the relationship of promoter ^(5m)CpG demethylation and geneexpression from the results of KEGG pathway analysis, the expression ofkey genes (DUSP7,8 and MAPK1) and their target genes CREB5, PRDX6, andBAD in the MAPK pathway, as well as the key genes CACNA1D (CaV1),CACNA1A (CaV2), and CACNA1H (CaV3) (encoding for calcium voltage-gatedchannel subunits) and their targeting genes (PGC1A, HMGR, and FAS) inthe calcium-AMK pathway were determined by RT-PCR analysis. TheDUSP-MAPK signaling pathway is the major pathway involved in cellsurvival/death and anti-oxidization, and the calcium signaling pathwaycontrols lipid and energy metabolism. As expected, 25HC3S increasedexpression of DUSP8 by 5-fold and its targeting gene, CREB5, by up to20-fold, which is the key element involved in cell survival and death(FIGS. 3A and 3B). Meanwhile, 25HC3S treatment significantly increasedexpression of key genes involved in the calcium signaling pathway, andits down-stream element, PGC1A, by 12-fold, while it decreasedexpression of HMGR and FAS genes by ˜90%, which encode the key enzymescontrolling energy metabolism in mitochondria, cholesterol biosynthesis,and fatty acid biosynthesis, as shown in FIGS. 3C and 3D.

Transcriptional Array Analysis in Hepatocytes

To examine the effect of 25HC3S on whole gene expression in humanhepatocytes, human Genome U133Aplus2.0 Genechip® array analysis of38,500 full length genes and EST (expressed sequence tags) clustersshowed that treatment with 25HC3S in HpG-2 cells significantly modulatedmany clusters of gene expressions. The major clusters affected are genesinvolved in cholesterol and triglyceride metabolism, cell survival, andinflammation. Genes associated with cholesterol and triglyceridebiosynthesis were significantly down-regulated, while genes associatedwith cell survival, proliferation, and anti-oxidization weresignificantly upregulated as shown in FIG. 4 . Altogether, 25HC3Smodulated the transcription of 1,276 genes (>1.6 fold) in atime-dependent manner. Genetic analysis of different GO processes, acollection of genes associated with a specific biological functionalprocess, revealed that at 8 hours, the majority of up-regulated pathwaysare involved in cell survival (FIGS. 4A and B); in contrast, majority ofdown-regulated genes are involved in lipid metabolism (FIGS. 4C and D).The up-regulated genes related with anti-apoptosis (increased by 3 to12-fold at 8 hours) are listed in FIG. 4E; and the down-regulated genesrelated with lipid metabolism (decreased by 50% to 95%) are listed inFIG. 4F. The detailed individual up-regulated genes are listed in Table1.7; the down-regulated genes are listed in Table 1.8. Many studies haveshown that epigenetic modification could globally regulate geneexpression involved in vital cellular functions, including metabolism,inflammation, and cell death/proliferation. Our data demonstrates that25HC3S epigenetically regulates gene expressions via DNA ^(5m)CpGdemethylation in promoter regions.

TABLE 1.7 Up Regulated Gene List of Huh-7 Cells Treated by 25HC3S for 8hours. Gene Symbol Fold Change Gene Name or Function IL8 11.91Interleukin 8 ANKRD1 8.67 Ankyrin Repeat Domain 1 FSTL3 8.24 Follistatinlike 3 CYR61 8.08 Cysteine rich angiogenic inducer 61 EDN1 8.03Endothelin 1 C11orf96 6.30 Description: chromosome 11 open reading frame96 BIRC3 5.81 Baculoviral IAP repeat containing 3 IL11 5.53 Interleukin11 HBEGF 4.43 Heparin binding EGF like growth factor CYP24A1 4.33Cytochrome P450 family 24 subfamily A member 1 SERPINE1 4.20 Serpinfamily E member 1 DDIT3 4.10 DNA damage inducible transcript 3 ATF3 4.08Activating transcription factor 3 HSPA6 3.92 Heat shock protein family A(Hsp70) member 6 TNS1 3.89 Tensin 1 DUSP1 3.88 Dual specificityphosphatase 1 KLF5 3.88 Kruppel like factor 5 THBS1 3.82 Thrombospondin1 SLC2A14 3.73 Solute carrier family 2 member 14 PMAIP1 3.65Phorbol-12-myristate-13-acetate-induced protein 1 CXCL2 3.63 Chemokine(C-X-C motif) ligand 2 KRTAP3-1 3.49 Keratin associated protein 3-1 SKIL3.36 SKI like proto-oncogene AKAP12 3.30 A-kinase anchoring protein 12TCIM 3.29 Transcriptional and immune response regulator ICAM1 3.22Intercellular adhesion molecule 1 GABBR1 3.20 Gamma-aminobutyric acidtype B receptor subunit 1 UBASH3B 3.15 Ubiquitin associated and SH3domain containing B SOCS2 3.15 Suppressor of cytokine signaling 2 CREB53.12 cAMP responsive element binding protein 5

TABLE 1.8 Down Regulated Gene List of Huh-7 Cells Treated by 25HC3S for8 hours. Percentage Gene Symbol Change (%) Gene name or function SC4MOL−81.12 Methylsterol monooxygenase 1 SLCO4C1 −72.30 Solute carrierorganic anion transporter family member 4C1 HMGCR −71.323-hydroxy-3-methylglutaryl-CoA reductase PNPLA3 −70.45 Patatin likephospholipase domain containing 3 ANKRD43 −69.19 Sosondowah ankyrinrepeat domain family member A HMGCS1 −67.713-hydroxy-3-methylglutaryl-CoA synthase 1 TUBB8 −67.29 Tubulin beta 8class VIII IDI1 −66.48 Isopentenyl-diphosphate delta isomerase 1 TNFSF10−65.42 TNF superfamily member 10 NCMAP −65.26 Non-compact myelinassociated protein RHOBTB1 −64.85 Rho related BTB domain containing 1EHHADH −64.46 Enoyl-coa hydratase and 3-hydroxyacyl coa dehydrogenaseSQLE −64.42 Squalene epoxidase PCSK9 −62.51 Proprotein convertasesubtilisin/kexin type 9 KANK4 −61.28 KN motif and ankyrin repeat domains4 SPTLC3 −60.32 Serine palmitoyltransferase long chain base subunit 3PAQR8 −60.05 Progestin and adipoq receptor family member 8 RALGPS1−59.86 Ral GEF with PH domain and SH3 binding motif 1 MAP2K6 −59.78Mitogen-activated protein kinase kinase 6 ZNF385B −58.25 Zinc fingerprotein 385B PLPPR1 −57.91 Phospholipid phosphatase related 1 SEC16B−57.72 SEC16 homolog B, endoplasmic reticulum export factor ID3 −57.51Inhibitor of DNA binding 3, HLH protein VAV3 −57.09 Vav guaninenucleotide exchange factor 3 KLLN −56.31 Killin, p53 regulated DNAreplication inhibitor SCN1A −56.24 Sodium voltage-gated channel alphasubunit 1 PLA2G12B −56.10 Phospholipase A2 group XIIB FRMD3 −55.75 FERMdomain containing 3 ID4 −55.58 Inhibitor of DNA binding 4, HLH proteinSLCO2B1 −55.27 Solute carrier organic anion transporter family member2B1 KLB −54.22 Klotho beta FABP1 −54.20 Fatty acid binding protein 1SORBS2 −53.92 Sorbin and SH3 domain containing 2 POU2AF1 −53.59 POUclass 2 homeobox associating factor 1 METTL7A −53.26 Methyltransferaselike 7A RAB11FIP4 −53.16 RAB11 family interacting protein 4 MAT1A −53.04Methionine adenosyltransferase 1A CELSR2 −53.00 Cadherin EGF LAGseven-pass G-type receptor 2 AGTR1 −52.98 Angiotensin II receptor type 1ELOVL6 −52.72 ELOVL fatty acid elongase 6 MVK −52.63 Mevalonate kinaseCYB5B −52.60 Cytochrome b5 type B CYP51A1 −52.40 Cytochrome P450 family51 subfamily A member 1 FDFT1 −52.07 Farnesyl-diphosphatefarnesyltransferase 1 PRLR −51.88 Prolactin receptor ALAD −51.76Aminolevulinate dehydratase PAQR9 −51.51 Progestin and adipoq receptorfamily member 9 FMC1 −51.27 Formation of mitochondrial complex Vassembly factor 1 homolog P2RY8 −50.91 P2Y receptor family member 8 TAB3−50.37 TGF-beta activated kinase 1 (MAP3K7) binding protein 3 ADH6−50.18 Alcohol dehydrogenase 6 (class V) NAP1L5 −50.17 Nucleosomeassembly protein 1 like 5 TMEM170B −50.02 Transmembrane protein 170B

The Calcium, AMPK, and PPAR signaling pathways are ones involved inregulation of energy, lipids, and carbohydrate metabolisms. TheCa²⁺/calmodulin-dependent protein kinase (CaMKK) and AMPK signalingpathway increases expression and decreases acetylation of PGC-la, whichregulates mitochondrial biogenesis and lipid metabolism. The data shownin Example 1 from analysis of Whole Genome-Wide DNA Methylation (genomiclevel) and transcriptional Array of Human Genome U133Aplus2.0 Genechip®(mRNA level) showed that 25HC3S treatment significantly demethylated^(5m)CpG in the promoter regions of key genes including calciumchannels, as well as genes of CaMKK and AMPK, increased theirexpression, and modulated downstream elements. These results providedevidence that 25HC3S globally regulated metabolic pathways mainly viathe Calcium-AMPK signaling pathway as shown in FIG. 5 . 25HC and 25HC3Sare potent modulators in regulating DNA methylation. 25HC methylatesCpG, and 25HC3S demethylates ^(5m)CpG, while also down- andup-regulating expression of the key genes. PGC-1α is a key regulator ofmitochondrial biogenesis, oxidative phosphorylation, and mitochondrialantioxidant defense, and it is also responsible for maintainingmetabolic homeostasis. PGC-1α expression is up-regulated by the CREBprotein and the AMPK signaling pathway. The present finding shows that25HC3S up-regulates expression of CREB and AMPK via demethylating^(5m)CpG in their promoter regions, and subsequently increasesintracellular PGC-1α levels (FIG. 3 ), which provides a detailedmechanism for how 25HC3S functions as proposed in FIG. 5 . 25HC3Ssuppresses DNMTs activities and demethylates ^(5m)CpG in the keypromoter regions. The demethylation up-regulates gene expression andincreases MAPK-CREB signalings, which blocks cell apoptosis, inducescell proliferation. The demethylation also up-regulates calcium-AMPKsignaling, resulting in inhibition of SREBP-1 activity by which inhibitsfatty acid and triglyceride biosynthesis, and inhibition of HMGCRexpression, decreases in cholesterol biosynthesis, and increases in thelevels of malnonyl-CoA as shown FIG. 5 .

Conclusion

The oxysterol sulfate, 25-hydroxycholesterol-3-sulfate (25HC3S) has beenshown in this example to play an important role in lipid metabolism,inflammatory response, and cell survival. Example 1 provides a study ofthe molecular mechanism by which 25HC3S functions as an endogenousepigenetic regulator. The kinetic study of epigenetic enzymesdemonstrated that 25HC3S specifically inhibited DNA methyltransferases,DNMT1, DNMT3a, and DNMT3b with IC50=4.04, 3.03, and 9.05×10-6 M,respectively. In human hepatocytes, high glucose induces lipidaccumulation by increasing promoter CpG methylation of key genesinvolved in development of non-alcoholic fatty liver diseases (NAFLD).Using this model, 25HC3S converted the 5mCpG to CpG in the promoterregions of 1074 genes involved in 79 KEGG pathways. Expression of thedemethylated genes, which are involved in the master signaling pathways,including MAPK-ERK, calcium-AMPK, and type II diabetes mellituspathways, increased. Messenger RNA array analysis showed that theup-regulated genes encoding for key elements in keeping cell survivaland the down-regulated genes encoding for key enzymes in decreasinglipid biosynthesis. The results shown in Example 1 indicate that theexpression of these elements and enzymes are regulated by thedemethylated signaling pathways, and 25HC3S DNA demethylation of 5mCpGin promoter regions is a potent regulatory mechanism.

Example 2 Objective

The objectives of this study were to determine the plasmapharmacokinetics of 25HC3S-derived radioactivity in male Sprague Dawleyrats, determine the routes of elimination and excretion mass balance of[4-¹⁴C]-25HC3S-derived radioactivity in male Sprague Dawley rats,determine the tissue distribution and tissue pharmacokinetics of[4-¹⁴C]-25HC3S-derived radioactivity using quantitative whole bodyautoradiography methods in male Sprague Dawley and Long Evans ratsfollowing a single intravenous (bolus) dose, and to provide plasma,urine, and fecal homogenate samples for metabolite profiling of[4-¹⁴C]-25HC3S-derived radioactivity.

Study Design

Nine male Sprague Dawley rats (Group 1) were designated for thepharmacokinetic phase, 3 male Sprague Dawley rats (Group 2) for theexcretion mass balance phase, and 7 male Sprague Dawley rats (Group 3)and 9 male Long Evans rats (Group 4) for the tissue distribution phase.All animals received a single intravenous dose of [¹⁴C]-25HC3S at 10mg/kg and a target radioactivity of 225 μCi/kg. Blood samples werecollected from all Group 1 animals at approximately 0.083, 0.25, 0.5, 1,2, 4, 8, 12, 24, 48, and 72 hours post-dose. Urine and feces werecollected from all Group 2 animals periodically through 168 hourspost-dose. At approximately 0.083, 0.5, 1, 4, 8, 24, and 168 hourspost-dose for Group 3 and at approximately 0.083, 0.5, 1, 4, 8, 24, 168,336, and 504 hours post-dose for Group 4, 1 animal/group/time point wasanesthetized with isoflurane and a blood sample collected. Followingblood collection, animals were euthanized by CO₂ inhalation andcarcasses frozen in a dry ice/hexane bath for processing by quantitativewhole body autoradiography. Whole blood, plasma, urine, feces, cagerinse, and cage wash were analyzed for total radioactivity by liquidscintillation counting.

Results and Key Findings

After a single intravenous (bolus) dose of [4-¹⁴C]-25HC3S administeredto rats at 10 mg/kg, the mean plasma C₀ was 25,900 ng-equiv./g, andAUC_(last) was 27,900 h*ng-equiv./g. The terminal elimination phaseT_(1/2) was 26.6 hours.

Based on the excretion data, approximately 100.2% of the doseadministered was recovered over 168 hours in urine, feces, and cagerinse from rats following a single intravenous (bolus) dose of[4-¹⁴C]-25HC3S at 10 mg/kg. The majority of the recovered radioactivitywas in feces (83.0%), indicating that biliary excretion is the primaryroute of excretion in rats.

After a single intravenous (bolus) dose of [4-¹⁴C]-25HC3S to maleSprague Dawley rats in Group 3 at 10 mg/kg, [4-¹⁴C]-25HC3S and/or itsmetabolites were broadly distributed and detected by quantitative wholebody autoradiography in all tissues except the eye (lens). Plasmaconcentrations were similar to those determined in the pharmacokineticsphase. The whole blood C_(max) was 8530 ng-equiv/g, and AUC_(last) was25,200 h*ng-equiv./g. There was a negligible difference in plasma andwhole blood exposure, as measured by the plasma:whole blood AUC_(last)ratio of 0.79, indicating that the 25HC3S partitioned equally intoplasma and blood cells. The T_(1/2) was 44.3 hours in plasma and 52.2hours in whole blood; differences in plasma T_(1/2) between the PK phaseand the QWBA phase are due to the difference in blood collection timepoints.

The C_(max) and AUC_(last) for [4-¹⁴C]-25HC3S-derived radioactivity werehighest in the liver: up to 87,900 ng-equiv./g and 364,000 h ng/g,respectively. Kidney (all sections), small intestine (wall), lung, andadrenal gland concentrations ranged from 43,200 ng-equiv./g to 13,600ng-equiv./g, higher than the maximum plasma concentration of 12,400ng-equiv./g. Thymus, bone (femur), uveal tract, fat, testes, and brainconcentrations were lowest relative to the other tissues: <5000ng-equiv./g (around 1500 ng-equiv./g). Remaining tissues hadconcentrations between 5000 and 10,800 ng-equiv./g. The T_(max) was mostoften 0.083 to 0.5 hours post-dose. Concentrations were belowquantitation limit in all tissues except adrenal gland, harderian gland,liver, and small intestine by 168 hours post-dose. As calculated usingAUC_(last), the tissue:plasma ratios were high for liver and smallintestine (wall) at 11.4 and 7.44, respectively. High liver and smallintestine concentrations are consistent with extensive biliary (fecal)excretion following an intravenous dose. All other tissue:plasma ratiosdemonstrated limited affinity for remaining tissue types.

Administration of a single intravenous dose of [4-¹⁴C]-25HC3S to maleLong Evans rats at 10 mg/kg revealed no substantial difference in plasmaor whole blood concentrations over the first 168 hours post-dose versusSprague Dawley rats; plasma and whole blood concentrations were belowquantitation limit in plasma and whole blood by 336 hours post-dose inpigmented animals. There appeared to be no difference in binding topigmented or non-pigmented skin or the uveal tract; for all tissues, theconcentrations were below quantitation limit by 168 hours post-dose.

Plasma, urine, and feces from rats were analyzed for determination of25HC3S related radiolabeled materials. Samples were profiled using highperformance liquid chromatography with radiodetection and metaboliccharacterization was performed using mass spectrometry and tandem massspectrometry analysis.

Plasma pools were made from Group 1 rats at the 0.083, 0.25, 0.5, and1-hour collection time points. From these Group 1 sample pools and froma Group 3 0.083-hour plasma sample, the largest component present in the0.083- and 0.25-hour collections was attributed to the parent 25HC3Srepresenting about 58% to 92% of the radioactivity. Three metabolitespresent at >10% of the radioactivity in the 0.5- and 1-hour collectionswere M14 (up to 15% relative observed intensity), M24 (up to 13%relative observed intensity), and M28 (up to 83% relative observedintensity). Among the time points with suitable radioactivity formetabolite profiling and characterization (up to 1 hour post-dose),approximately 54% of the exposure (AUC) to 25HC3S related radioactivitywas attributable to 25HC3S, approximately 34% to M28, and the remainderto the minor metabolites.

Urine pools were prepared for Group 2 at 0 to 6 and 6 to 12 hourspost-dose. The largest component present was attributed to the parent25HC3S representing about 78% to 93% of the radioactivity. A total of 4metabolites were identified, although no metabolites were presentat >1.2% of dose or >10% relative observed intensity. Four metabolitespresent at <10% relative observed intensity in at least 1 sample were M7(<5% relative observed intensity), M16 (<3% relative observedintensity), M19 (<6% relative observed intensity), and M25 (<5% relativeobserved intensity).

Feces pools were prepared for Group 2 at 0 to 12, 12 to 24, and 24 to 48hours post-dose.

A total of fourteen metabolites were identified. Four metabolitespresent at ≥5% of dose were M1 (21% of dose and 23% to 30% relativeobserved intensity), M2 (7% of dose and 4% to 12% relative observedintensity), M3 (15% of dose and 13% to 23% relative observed intensity),and M4 (8% of dose and 6% to 12% relative observed intensity). Parent25HC3S was present at 2% of dose (1% to 5% relative observed intensity).

The primary metabolic pathways involved oxidation of 25HC3S resulting inthe conversion of the sulfate group to a hydroxyl group followed byfurther oxidation to form bile acid structures related to deoxycholicacid and cholic acid or their isomers. In addition, glutathioneconjugation of deoxycholic acid (or an isomer of deoxycholic acid) wassuggested by the presence of a metabolite having the correspondingmolecular weight for that structure. Neither desmosterol sulfate nor25-hydroxycholesterol was detected in any of the plasma, urine, or fecessamples.

Example 3

After a single oral (gavage) dose of [¹⁴C]-25HC3S administered to ratsat 75 mg/kg, plasma C_(max) was 3800 ng equiv./g, and AUC_(last) was96,400 h·ng equiv./g. The terminal elimination phase T_(1/2) was 27.3hours.

Based on the excretion data, approximately 94.5% of the doseadministered was recovered in urine, feces, and cage rinse from ratsfollowing a single oral (gavage) dose [¹⁴C]-25HC3S at 75 mg/kg. Themajority of the recovered radioactivity was in feces (94.2%), indicatingthat biliary excretion is the primary route of excretion for absorbed25HC3S in rats.

After a single oral (gavage) dose of [¹⁴C]-25HC3S to male Sprague Dawleyrats at 75 mg/kg, [¹⁴C]-25HC3S and/or its metabolites were broadlydistributed and detected by quantitative whole body autoradiography inall tissues except the eye (lens). No [¹⁴C]-25HC3S-derived radioactivitywas detected in the eye (lens). Plasma concentrations were similar tothose determined in the pharmacokinetics phase and were above the lowerlimit of quantitation. The whole blood C_(max) was 2850 ng equiv/g, andAUC_(last) was 127,000 h ng equiv./g. There was a negligible differencein plasma and whole blood exposure, as measured by the plasma:wholeblood AUC_(last) ratio of 1.12, indicating that the 25HC3S partitionedapproximately equally into plasma and blood cells.

For the tissues analyzed by quantitative whole-body autoradiography, theC_(max) for [¹⁴-C]-25HC3S-derived radioactivity, where measurable, washighest in the small intestine (wall) followed by the stomach (wall):424,000 ng equiv./g and 204,000 ng equiv./g, respectively. Pancreas andliver concentrations ranged from 23,500 ng equiv./g to 28,100 ngequiv./g. Uveal tract and brain concentrations were lowest relative tothe other tissues and were approximately 1000 ng equiv./g. Skin, thymus,prostate, and pituitary tissue concentrations were <3000 ng equiv./g.Remaining tissues had concentrations between 3600 ng-equiv./g and 10,700ng equiv./g. The T_(max) was 6 hours post-dose or less. By 168 hourspost-dose, tissue concentrations were near or below the quantitationlimit in all tissues except adrenal gland and liver. As calculated usingAUC_(last), the tissue:plasma ratios were highest for the smallintestine (wall, 15.4) followed by the liver and adrenal gland at 6.96and 6.64, respectively. High liver and small intestine concentrationsare consistent with oral administration and biliary (fecal) excretion.All other tissue:plasma ratios demonstrated limited affinity forremaining tissue types.

Radiolabeled components in plasma and feces extracts were profiled andidentified using radio-high performance liquid chromatography (HPLC) andhigh performance liquid chromatography/mass spectrometry (HPLC/MS)methods.

There were no urine samples that contained sufficient radioactivity torequire metabolite profiling and identification.

Plasma pools were prepared for Group 1 (75 mg/kg, [¹⁴C]-25HC3S) samplescollected at 2, 4, and 6 hours post-dose. In the 2 hour post-doseplasma, the primary radiolabeled component was parent 25HC3S which waspresent at 63% relative observed intensity (ROI) and a concentration of2090 ng-equiv./g. One metabolite M29 was identified as25-hydroxycholesterol with 37% ROI and a concentration of 1233ng-equiv./g. The plasma collections at 4 and 6 hours post-dose did notcontain sufficient concentrations for radioprofiling.

Feces pools were prepared for Group 2 (75 mg/kg, [¹⁴C]-25HC3S) samplescollected from 0 to 24, 24 to 48, 48 to 72, 72 to 96, 96 to 120, 120 to144, and 144 to 168 hours post-dose. A total of eleven metabolites wereidentified. None of the metabolites were present at ≥5% of dose.Metabolites present at 2-5% of dose were M1 (4.5% of total dose and1%-69% ROI), M3 (4.6% of total dose and 1%-44% ROI), M4 (2.0% of totaldose and 0%-10% ROI), M8 (3.1% of total dose and 1%-46% ROI), M29 (1.9%of total dose and 0%-2% ROI), and M30 (3.3% of total dose and 0%-5%ROI). The primary radiolabeled component was parent 25HC3S which waspresent at 71.1% of total dose (0%-88% ROI).

Radiolabeled desmosterol sulfate was not found in any of the plasma orfeces samples.

The primary metabolic pathways involved oxidation of 25HC3S, resultingin the conversion of the sulfate group to a hydroxyl group followed byfurther oxidation to form bile acid structures related to deoxycholicacid and cholic acid or their isomers and 25-hydroxycholesterol.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. Moreover, nothing disclosedherein is intended to be dedicated to the public regardless of whethersuch disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to belimited to the exemplary embodiments shown and described herein. Rather,the scope and spirit of present invention is embodied by the appendedclaims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) isexpressly defined as being invoked for a feature in the claim only whenthe exact phrase “means for” or the exact phrase “step for” is recitedat the beginning of such feature in the claim; if such exact phrase isnot used in a feature in the claim, then 35 U.S.C. § 112(f) or 35 U.S.C.§ 112(6) is not invoked.

1. A method of treating at least one of depression, neurodegenerativedisease, multiple sclerosis, Parkinson's disease, spinocerebellardegeneration, Friedreich ataxia, ataxia-telangiectasia, progressivesupranuclear palsy, Huntington's disease, striatonigral degeneration,olivopontocerebellar atrophy, Shy-Drager syndrome, schizophrenia,schizoaffective disorder, manic-depression (bipolar) disorder, disturbedor abnormal circadian entrainment, childhood Alice in Wonderlandsyndrome, childhood acute cerebellar ataxia, and Alzheimer's disease, ina subject in need thereof, comprising: administering to the subject aneffective amount of at least one compound selected from25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-expoxycholesterol-3-sulfate, or salt thereof.
 2. A method oftreating an addiction in a subject in need thereof, the methodcomprising administering to the subject at least one compound selectedfrom 25-hydroxycholesterol-3-sulfate (25HC3S),25-hydroxycholesterol-disulfate (25HCDS),27-hydroxycholesterol-3-sulfate (27HC3S),27-hydroxycholesterol-disulfate (27HCDS),24-hydroxycholesterol-3-sulfate (24HC3S),24-hydroxycholesterol-disulfate (24HCDS), and24,25-epoxycholesterol-3-sulfate, or salt thereof in an amount effectiveto reduce the addiction.
 3. The method of claim 2, wherein the addictionis at least one of drug addiction and behavioral addiction.
 4. Themethod of claim 2 or 3, wherein the addiction comprises drug addictionor wherein the addiction comprises addiction to a drug comprising atleast one of alcohol, opioid, cocaine, and amphetamine.
 5. The method ofclaim 4, wherein administering the at least one compound is effective toreduce one or more of: a physiological dependence on the drug by thesubject; a psychological dependence on the drug by the subject; andcravings for the drug by the subject.
 6. The method of any one of claims1 to 5, wherein the method comprises administering to the subject aneffective amount of 25-hydroxycholesterol-3-sulfate (25HC3S) or saltthereof.
 7. The method of any one of claims 1 to 6, wherein the at leastone compound is administered in an amount selected from the groupconsisting of: (a) an amount ranging from 0.001 mg/kg/day to 100mg/kg/day; (b) an amount ranging from 0.1 mg/kg to 100 mg/kg, based onbody mass of the subject; and (c) an amount ranging from 1 mg/kg to 10mg/kg, based on body mass of the subject.
 8. The method of any one ofclaims 1 to 7, wherein the administering is performed from once to 3times per day.
 9. The method of any one of claims 1 to 8, wherein theadministering comprises at least one of oral administration, entericadministration, sublingual administration, transdermal administration,intravenous administration, peritoneal administration, parenteraladministration, administration by injection, subcutaneous injection, andintramuscular injection.
 10. The method of any one of claims 1 to 9,wherein the administering comprises administering a pharmaceuticalcomposition comprising the at least one compound and a physiologicallyacceptable excipient, diluent, or carrier.
 11. The method of claim 10,wherein the pharmaceutical composition is formulated in unit dosageform.
 12. The method of claim 10 or 11, wherein the pharmaceuticalcomposition is in solid form.
 13. The method of any one of claims 10 to12, wherein the pharmaceutical composition: (a) is in the form of apowder, a tablet, a capsule, or a lozenge; and/or (b) comprises the atleast one compound in freeze-dried form together with a bulking agent.14. The method of claim 10 or 11, wherein the pharmaceutical compositioncomprises a carrier that is a liquid.
 15. The method of claim 14,wherein the at least one compound is solubilized in the liquid ordispersed in the liquid.
 16. The method of claim 14 or 15, wherein: (a)the liquid is aqueous; or (b) the liquid is sterile water for injectionsor phosphate-buffered saline.
 17. The method of any one of claims 10 and14 to 16, wherein the pharmaceutical composition is in a sealed vial,ampoule, syringe, or bag.